The antibacterial efficacy of intracanal medication with calcium hydroxide [Ca(OH)2], 2% chlorhexidine gel (CHX), and a combination of both [Ca(OH)2/CHX] was assessed in teeth with chronic apical periodontitis. Thirty-three canals were instrumented, randomly divided into three groups, and medicated with either Ca(OH)2, CHX, or Ca(OH)2/CHX. Bacteriological samples obtained from the operative field and the root canals before (S1) and after instrumentation (S2) in the first treatment session, and after medication (S3) in the second session 1 week later, were assessed for bacterial growth, observed by turbidity and in agar plates, and viable colony-forming unit (CFU) counts. Bacterial growth and CFU counts decreased significantly from S1 to S2 (Mann-Whitney, p<0.05). Differences in growth and counts between S2 to S3 were not statistically significant for all three intracanal medication groups. It was concluded that the antibacterial efficacy of Ca(OH)2, CHX, and Ca(OH)2/CHX was comparable.