Aim: To compare the antimicrobial efficacy of sodium hypochlorite (NaClO at 2.5% and 5.25%) and calcium hypochlorite [Ca(ClO)2 at 2.5%] on a biofilm of Enterococcus faecalis ATCC 29212™ and Candida albicans ATCC 10231™. Materials and methods: We performed an experimental in vitro study. Strains of C. albicans and E. faecalis, which had previously been reactivated were used. Then the colonies to be used were standardized in a turbidity standard to guarantee a quantity of 108 (CFU/mL) using the McFarland scale (0.5). Subsequently, the biofilm formed in brain-heart infusion agar was seeded into 42 sterile disks previously embedded with the experimental substances. Both 2.5% NaClO and Ca(ClO)2 solutions were placed in each Petri dish. They were then incubated at 37°C for 24 hours and the inhibition halos were measured using the Kirby-Bauer technique. Results: The means between the halos corresponding to NaClO and Ca(ClO)2 at 2.5% were 13.38 ± 0.64 mm and 13.42 ± 0.62 mm, respectively. According to the Tukey test, no statistically significant differences were found between the hypochlorite groups evaluated (p = 0.989). Conclusion: Both Ca(ClO)2 and NaClO have a similar antimicrobial efficacy with biofilm based on E. faecalis and C. albicans, with no statistically significant differences between the two. Clinical significance: This study demonstrates the effectiveness of Ca(ClO)2 and NaClO as endodontic irrigators to combat the most frequent microorganisms of the root canal.