This study was aimed at comparing the cultivable microorganisms in canals with periapical radiolucencies with exposed and unexposed pulp space. Microbiological samples were taken and analyzed from 45 canals with exposed pulp space, and 43 canals with unexposed pulp space. The canal contents were analyzed by aerobic/anaerobic culture, and conventional identification techniques. There were 211 isolates of bacteria belonging to 28 genera and 55 species recovered from exposed canals. In the unexposed group, 185 isolates of bacteria were recovered, of which 54 species of 28 genera were identified. Among the four most common genera, Prevotella was significantly more common in the exposed group (51/211 in the exposed group versus 30/185 in the unexposed group) (p = 0.049), while there were no differences in prevalence of Actinomyces, Peptostreptococcus, and Campylobacter between two groups of canals. In addition, Fusobacterium nucleatum and Propionibacterium acne were significantly more common in the unexposed canals (p = 0.047 and p = 0.0051, respectively). Similarity in bacterial species in these two groups suggests that pulp space exposure may not be a significant factor in determining the type of bacteria present in infected canals.