Don John's importance in this comedy lies in the way that he is the source of many of the play's confusions that threaten to make this comedy into a tragedy. It is he who, even before the play has begun, has rebelled unsuccessfully against his brother, Don Pedro. His character as Don Pedro's bastard brother to Shakespeare's audience would have indicated that he was a n'er-do-well, as illegitimate children were associated with discord and mischief. Note what Don John says about himself in Act I scene 3:
...it must not be denied
but I am a plain-dealing villain.
Don John is therefore a self-confessed wicked character who, in spite of his brother's magnanimous nature and benificence is determined to do anything he can to attack and destroy the happiness of the rest of the characters. It is his determination to sow discord amongst the characters that results in Hero's reputation being sullied. This of course necessitates her "rebirth" in order for her to clear her name. Don John, through his evil plan, explicitly foregrounds one of the central themes of the play, which is the extremely fragile nature of a woman's reputation and how easily it can be tainted.
For any Shakespearian comedy, there is a thin dividing line that prevents it from falling into the category of a tragedy. Don John is the central character who works towards making this a tragic play, and his importance lies in the way that he attempts to turn a light-hearted romantic comedy into something much more sinister and alarming.