Abstract The aim of this study was to evaluate the effect of nisin, an antimicrobial peptide, on the development of plaque and gingivitis in beagle dogs when compared with 0.12% chlorhexidine and a placebo. 16 female beagle dogs 1 year of age were brought to optimum gingival health by scaling, root planing and polishing. At the conclusion of the pretreatment phase, the dogs were divided into 4 groups for the application of the test agents and were placed on a plaque promoting diet of Purina Dog Chow softened with water. Test agents included 100 μg/ml nisin and 300 μg/ml nisin formulated in a vehicle containing 1 mM NaEDTA; negative control comprised of exactly the same formulation but omitting nisin; and 0.12% chlorhexidine as Peridex®. Throughout the treatment period, formulations were applied 2 × daily to premolar teeth in each quadrant for 1 min using a Monojet syringe. The development of plaque and gingivitis was monitored at 15, 27, 39, 53, 74 and 88 days during the treatment phase using standard measurements of gingival index, plaque index, stain index and bleeding to probing. Throughout the treatment phase, plaque accumulation increased in all groups, but the rate of plaque build-up was less in groups treated with either nisin or chlorhexidine formulations compared with the placebo treated group. The gingival index of dogs in all groups increased throughout the study period. However, from day 27 onward, the groups receiving nisin had lower gingival index scores than did the placebo group. At day 39 and onward, the chlorhexidine group had significantly lower gingival index scores than the placebo group. All groups demonstrated an increased in % Weeding sites. However, from day 27 onward, the 300, μg/ml nisin group and from day 39 the 100 μg/ml group, had significantly fewer % bleeding sites than the placebo group. An evaluation of the staining intensity index revealed that staining for the chlorhexidine-treated group was significantly greater than that of placebo and nisin at days 27, 74 and 88. In conclusion, nisin is an antimicrobial agent and is effective in the reduction of plaque build-up and gingivitis in the beagle dogs.