Unrest had been simmering among shirtwaist workers for a long time, and in November 1909, a group called Local 25 called a meeting to discuss a general strike. Samuel Gompers, the most powerful union leader in the United States, preached caution alongside other high-profile speakers. Clara Lemlich, unsatisfied with the speakers' advice to hold off and wait, stood and declared a strike with a rousing speech, feeling that so far the meeting had been "talk-talk that meant nothing." After being invited to the podium, the audience unanimously voted for a strike, and the next day an estimated 15,000 shirtwaist workers walked out of factories.

The turnout was unprecedented, and the Local 25 and WTUL frantically rented more meeting places to allow the striking workers to convene. The demands of the strike included a twenty percent pay raise, a fifty-two-hour workweek, and a closed shop system—meaning that the union would be recognized as the bargaining agent for all shirtwaist workers. As well as this, strikers wanted clear rules and extra pay for overtime and busy seasons, and advance notice when there would be no work to do.

The strike was divided along ethnic lines, and factory owners tried to take advantage of this. The strikers, who were predominantly Jewish, came from a community that preached unionism and the importance of worker solidarity. Italians, who largely joined the strikebreakers' side of the conflict, "were very much afraid of trade unions," according to Mary Dreier, a WTUL member and union executive. Dreier worried that the factory owners would try to incite "race warfare," and measures were taken to try to convince Italian workers to join the side of the strikers, including house visits from WTUL volunteers. An important moment in the strike was the cutters' union's decision to join; cutters were the best-paid workers, all men, very skilled and thus difficult to replace. Within the first forty-eight hours, more than seventy owners—around one in seven—had surrendered, all proprietors of smaller shops who would be unable to sustain closure for a long period of time.

While these workers immediately returned to work, owners of large factories, including Blanck and Harris, convened an emergency meeting and agreed that the strike must be resisted. They formed a manufacturers' association, the Allied Waist and Dress Manufacturers Association, and vowed not to grant a single union demand. The day after the meeting, Blanck and Harris tried to keep as many workers on the job as possible, inviting the eighth-floor workers up to the ninth floor at lunch and playing music for people to dance to. Everyone "peeled oranges and ate rolls and sipped tea," and Blanck and Harris hoped this would entice the workers away from joining the strike. Simultaneously, factory management hardened their position and picket lines became more violent, with police officers using increasingly brutal techniques to break the strike. The arrest of female strikers became an important part of the movement, as it caught the attention and therefore the support of upper-class socialites.

Strikers Rose Perr and Annie Albert were arrested after deciding to talk to the strikebreakers and "ask them why they work, and tell them we are not going to harm them at all." As they walked forward, a tall man punched Albert in the chest, and Perr yelled for help from the police. Both Albert and Perr were subsequently arrested and sent to the Jefferson Market magistrate's court. Violet Pike of the WTUL posted their bail, but Albert and Perr were ultimately sentenced to five days hard labor. Their sentences and the strike caught the attention of upper-class women such as Anne Morgan, the daughter of Wall Street financier J. P. Morgan. Anne Morgan and other likeminded women held a meeting to support the workers and organized a collection to fund the strike.

The socialites and heiresses had turned the shirtwaist strikers' movement into a politicized moment for women's rights, arguing that unfair sentencing and poor working conditions would not be a problem if women were allowed the vote. Although the upper-class supporters preached solidarity between women of all classes in the movement, there was significant resentment toward them from the striking workers. Many working-class women thought it unfair that the heiresses received the greatest share of media attention, and did not trust that upper-class women could truly sympathize with the workers’ experiences.

Amid the worst blizzard New York had seen in twenty years, Local 25 rejected a mediation proposal from manufacturers and refused to discuss anything less than full union recognition and a closed shop system. Many progressives who sympathized with the cause held the view that the refusal to negotiate was too radical, as closed shop systems were seen as at the time. Suspicions of radicalism came to a head on January 2nd, 1910, when strikers and wealthy supporters alike crowded into Carnegie Hall and listened to speeches about workers' rights and the shirtwaist strike. A rousing speech by Attorney Morris Hillquit and the WTUL's immediate defense of it was condemned by Anne Morgan and other rich supporters, resulting in a breakdown of the strikers’ apparent solidarity.

The strike came to a close in February 1910, when the WTUL and Local 25 essentially accepted the deal they had previously rejected. Blanck and Harris refused to back down, and most of the shops took strikers back on higher wages and shorter hours; they recognized the union, but only in the sense that they no longer prohibited membership. Thus, although "strike supporters tried to present the result as a victory," Blanck and Harris had successfully prevented a closed shop system and in fact represented violent resistance to the strike rather than acceptance of its participants’ demands.