The aim of this study was to evaluate the antibacterial potential of a calcium silicate-based sealer (Bio-C Sealer, Angelus) against common bacteria in primary and secondary endodontic infections. Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus mutans were exposed to fresh Bio-C Sealer for 24 h by the agar diffusion method (n=5). Additionally, the antibacterial activity was investigated against E. faecalis and S. mutans biofilms (48 h old) grown in discs with 4 mm in diameter and 2 mm in height. (n=3) of set discs of Bio-C Sealer (Angelus), EndoFill (Dentsply-Mallefer), Sealer 26 (Dentsply), AH Plus (Dentsply), Sealapex (Sybron-Endo) and EndoSequence BC Sealer (Brasseler). The antibacterial activity was evaluated by colony forming unity (CFU) counting using ImageJ software. Data were compared by one-way ANOVA followed by Holm-Sidak test (a=5%). Fresh Bio-C Sealer exhibited antimicrobial activity against all bacteria evaluated by agar diffusion method, except for S. mutans. Set discs of all endodontic sealers tested showed similar CFU values for E. faecalis (p>0.05). S. mutans in biofilms showed higher susceptibility to EndoFill compared with the other sealers (p<0.05). In conclusion, the results indicate that fresh Bio-C Sealer does not inhibit S. mutans growth, but exhibits antibacterial activity against E. faecalis, S. aureus, P. aeruginosa and E. coli. After setting, the Bio-C Sealer exhibits an antimicrobial potential comparable to that of the other sealers evaluated in E. faecalis biofilm, but lower than that of EndoFill for S. mutans biofilm.