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Context: The present study was aimed to explore new irrigating solutions, which would probably be as effective as sodium hypochlorite. Aims: The aim of this in vitro study was to evaluate the antibacterial effectiveness of 0.1% octenidine, SmearOFF, 1% alexidine and 5.25% sodium hypochlorite against E. faecalis biofilm formed on tooth substrate. Materials and methods: Eighty extracted human teeth were decoronated, biomechanically prepared, vertically sectioned, placed in the tissue culture wells exposing the root canal surface to E. faecalis strain (ATCC 29212) to establish 3-week-old biofilm. At the end of the 3rd week all groups were treated for 10 minutes with 3 ml of test solutions. Samples exposed to 5.25% sodium hypochlorite (NaOCl) were used as control. All the samples were evaluated for E. faecalis growth and number of colony forming units. Statistical analysis was performed by using one-way analysis of variance (ANOVA) to measure the mean values. The intergroup comparison was done by Tukey HSD post hoc test (p<0.05). Results: Maximum inhibition was observed with 0.1% OCT (group 1) followed by NaOCl (group 4) and ALX (group 3). SmearOFF (group 2) showed the least inhibitory effect. NaOCl (group 4) and ALX (group 3) showed no statistically significant difference (p>0.05), whereas all the other intergroup differences were statistically significant (p<0.05). Conclusions: 0.1% OCT showed significantly higher antibiofilm effects. No significant differences were observed between 1% ALX and 5.25% NaOCl. SmearOFF showed significantly lower antibiofilm effects (p<0.05). [ABSTRACT FROM AUTHOR]