BACKGROUND Biofilm makes bacteria resistant to antimicrobial agents and facilitates the transmission of infectious diseases in hospitals. Disinfectant compounds are frequently used to control surface contamination. This study was designed to investigate the effect of chlorhexidine (CHX) and hydrogen peroxide (H2O2) on biofilm formation of Enterococcus faecalis. METHODS This study was performed on 40 E. faecalis clinical isolates. After the determination of MIC, the effect of different concentrations of CHX and H2O2 on the biofilm formation was evaluated. Also, the relative expression level of the studied biofilm genes, following exposure to sublethal concentration of CHX and H2O2, was assessed using quantitative reverse transcription PCR (qRT-PCR). RESULTS The frequency of the asa1, efaA, epaI, and esp biofilm genes were 80%, 92.5%, 100%, and 75%, respectively. Various concentrations of CHX increased the biofilm mass in E. faecalis. Also, the combination of CHX and H2O2 at sub-minimal inhibitory concentrations, significantly elevated the expression of asa1, epaI, and esp genes. CONCLUSIONS The results of this study showed that the improper use of disinfectants can increase the ability of biofilm formation in E. faecalis and may cause selective pressure leading to the emergence of biocide-resistant microorganisms.