The aim of this study was to investigate in vitro the antimicrobial activity of calcium hydroxide [Ca(OH)2] in combination with different vehicles against endodontic pathogens. For such purpose, a broth dilution test was performed. Pastes were prepared with Ca(OH)2 powder and the following vehicles: sterile water, glycerin, camphorated monochlorophenol (CMCP), CMCP + glycerin, polyethyleneglycol and CMCP + polyethyleneglycol. The time required for the pastes to produce negative cultures against the tested microorganisms was recorded and analyzed statistically using the Kruskal Wallis test at 5% significance level. Timing for pastes to eliminate the aerobic and facultative anaerobic microorganisms ranged from 6 to 24 h, while strict anaerobic microorganisms were inhibited within 30 s to 5 min. Microbial susceptibility, ranked from weakest to strongest, can be presented as follows: Enterococcus faecalis (the most resistant microorganism), Candida albicans, Staphylococcus aureus, Porphyromonas gingivalis, Porphyromonas endodontalis and Prevotella intermedia (the last two microorganisms required the same time to be eliminated). In conclusion, calcium hydroxide pastes needed more time to eliminate facultative than anaerobic microorganisms. These findings suggest that the antimicrobial property is related both to paste formulation and to microbial susceptibility.