Objective: Present study assess the prevalence of microorganisms and their species in infected root canals of teeth which is one of major causes of pulpal and periapical diseases. Materials & methods: study conducted on 45 patients with involved teeth individually isolated with a disinfected rubber dam. Root canal was instrumented and samples were collected via paper points and inserted in the aerobic and anaerobic BACTEC 9050 bottles. In microbiology laboratory samples were incubated in BACTEC 9050 automated culture instrument for 24 hours. Results: Sphingomonas Paucimobilis and Entrobacter Cloacae(97.78% ) were most commonly observed in 22.22% and 13.33% of the patients respectively. Gram positive bacteria in 15.56% while gram negative bacteria in 82.22%. Aerobic bacteria were present in 67.67% of patients while anaerobic bacteria were present in 31.11% of patient. Conclusion: Gram negative and aerobic microorganisms are highly prevalent in infected root canals of teeth.