The antimicrobial effectiveness of calcium hydroxide, camphorated paramonochlorophenol, and formocresol in root canals of extracted human teeth was compared. Canals in single-rooted teeth were enlarged and inoculated with Streptococcus mutans, Actinomyces viscosus, and Bacteroides gingivalis or Bacteroides fragilis. After treatment with a test agent and sealing and incubation for 1 hour, the canal contents were analyzed for the number of viable test bacteria and compared with that of inoculated teeth not treated with test agents. All test agents exhibited antimicrobial activity against all bacteria, with percent reductions in viable bacteria ranging from 64.3% to 100%. The combined data for Pulpdent paste and calcium hydroxide showed significantly higher antimicrobial activity than the combined data for camphorated paramonochlorophenol and formocresol for S. mutans and B. gingivalis or B. fragilis but showed no difference for A. viscosus.