We assessed the effect of different compositions and concentrations of two etidronate-containing irrigants on the antibacterial activity of sodium hypochlorite (SH) against Enterococcus faecalis and Candida albicans in vitro. Pure cultures of C. albicans and E. faecalis were isolated from root canal samples. The disc diffusion method was used to compare the antibacterial effect of pure SH and SH mixed with 9%, 15%, and 18% etidronate of two manufactures (dual rinse (DR); IsraDent (ID)) and EDTA. The pH and temperature of the solutions were measured immediately after mixing and within 40 min. The ANOVA revealed a significant influence of the type of irrigating solution on the C. albicans and E. faecalis inhibition zone diameters that ranged from 6.6 to 51.6 mm and from 6.4 to 12.4 mm, respectively. SH with DR 9% exhibited the highest effect against C. albicans. The antifungal activity of the other irrigants was SH = SH + DR15% = SH + DR18% = SH + ID9% > SH + EDTA > SH + ID15% > SH + ID18%. No significant differences in the anti-E. faecalis effect were revealed between the tested solutions except for the mixtures of SH and 15% and 18% ID, which exhibited no antiseptic effect. There was a strong positive correlation between antiseptic activity against both microorganisms and the pH values of the tested solutions. In conclusion, most etidronate formulations did not significantly hamper sodium hypochlorite activity against C. albicans and E. faecalis. The effect was concentration- and manufacturer-dependent.