This study was designed to evaluate the antimicrobial effectiveness of 25% citric acid, compared with 2.5% sodium hypochlorite, after time intervals of 5, 15, 30 and 60 min. Anaerobic bacterial species were isolated from infected root canals and categorized according to Gram stain as Gram-positive cocci, Gram-negative cocci, Gram-positive rods and Gram-negative rods. The experimental method used was similar to that proposed by the German Society for Hygiene and Microbiology (DGHM) with slight modifications. Testing revealed that citric acid does demonstrate antimicrobial properties against anaerobic bacteria, especially against cocci, but it is less effective than 2.5% sodium hypochlorite.