Prevention of dental caries is a key research area, and improvement of the pit and fissure sealants used for caries prevention has been of particular interest. This report describes results of incorporating a zwitterion, sulfobetaine methacrylate (SB), into photo-polymerized resin-based sealants to enhance resistance to cariogenic bacteria and protein adhesion. Varying amounts (1.5–5 wt%) of SB were incorporated into a resinbased sealant, and the flexural strength, wettability, depth of cure, protein adhesion, bacterial viability, and cell cytotoxicity of the resultant sealants were evaluated. The flexural strength decreased with the increasing SB content, but this decrease was statistically significant only for sealants containing ≥3 wt% SB. Incorporating a zwitterion led to a significant reduction in the water contact angle and protein adhesion. The colony-forming unit count showed a significant reduction in the bacterial viability of S. mutans, which was confirmed with microscopic imaging. Moreover, cell cytotoxicity analysis of SB-modified sealants using an L929 fibroblast showed a cytotoxicity comparable to that of an unmodified control, suggesting no adverse effects on the cellular metabolism upon SB introduction. Hence, we conclude that the addition of 1.5–3 wt% SB can significantly enhance the inherent ability of sealants to resist S. mutans adhesion and prevent dental caries.