This study attempted to incorporate the antibacterial peptide nisin into an etch-and-rinse dental adhesive to evaluate the antibacterial activity of the modified adhesive against Streptococcus mutans and the bond strength. Single Bond 2 was used as a negative control, and nisin was incorporated at 1%, 3%, and 5% (w/v). The antibacterial activity against S. mutans was evaluated using the film contact test, the agar diffusion test, XTT assays and confocal laser scanning microscopy (CLSM). The microtensile bond strength (μTBS) of the modified dental adhesive was also evaluated. The cured nisin-incorporated dental adhesive exhibited a significant inhibitory effect on the growth of S. mutans (P < 0.05), and the inhibitory effect was strengthened as the nisin concentration increased (P < 0.05). However, no significant differences in the agar diffusion test were found for the cured nisin-incorporated adhesives compared with the control group. Based on XTT results and CLSM images, the cured nisin-incorporated adhesive interfered with the adherence of S. mutans and the integrity of its biofilms (P < 0.05). Compared with the control group, the 1% nisin group did not exhibit a significant difference in μTBS (P > 0.05), whereas the 3% and 5% nisin groups displayed decreased bond strength (P < 0.05).