When the Zollverein was created, it specifically excluded Austria; which played heavily in the Austrian-Prussian Rivalry. Austria's industries were protected by high tariffs; and lower tariffs within the Zollverein was disastrous for the Austrian economy. Austria tried to destroy the Zollverein by inducing the southern German states to leave it, but failed. By 1853 all German states except Austria were members; and as businessmen and merchants grew rich, it became increasingly apparent that a united Germany would not include Austria. Prussia was the largest member state of the Zollverein, and therefore had a substantial advantage over Austria in their rivalry to control German affairs. Later, when Otto von Bismarck became Chancellor of Prussia, he managed to ally all the German states against Austria and thereby exclude it from the unified Germany.
After the creation of the Zollverein, the relations between Austria and Prussia became even more contentious than they had been.
The creation of the Zollverein was meant in large part to strengthen Prussia. Prussia was in competition with Austria for influence over the various German states that existed at the time. By creating the Zollverein, Prussia pulled the German states into a closer relationship with it and away from a relationship with Austria. This exacerbated tensions with Austria because Austria was worried about its loss of influence over the German states as those states gravitated towards Prussia.
In this way, the creation of the Zollverein made relations between Prussia and Austria more hostile and tense.