Generally, mechanical plaque control without chemical support is insufficient to prevent oral diseases, and an ongoing quest exists for new antimicrobials for use in oral healthcare. Chitosans are polycationic, naturally occurring antimicrobials that are rapidly finding their way into oral healthcare. In this study we determined the effects of pellicle treatment with chitosan on bacterial adhesion and growth. Chitosan caused a reduction in bacterial adhesion and was responsible for bacterial death upon contact compared with a buffer control. Exposure of adhering bacteria to a chitosan solution or a buffer control did not cause detachment, but the chitosan solution left a small proportion of the adhering bacteria alive. Growth after exposure to chitosan was similar to (Streptococcus mutans ATCC700610, Streptococcus oralis HM1, Streptococcus sobrinus HG1025) or less than (S. mutans NS, Actinomyces naeslundii HM1) that of the control, while biofilm viability after chitosan treatment was lower than that of the control, except for S. oralis HM1. Therefore, chitosan is a promising antimicrobial for use in oral healthcare.