The objective of the study was to evaluate the bacteria extruded apically during root canal preparation using hand and two rotary instrumentation techniques. The method include eighty freshly extracted mandibular premolars were mounted in bacteria collection apparatus. Root canals were contaminated with the pure culture of Enterococcus faecalis (ATCC 29212) and dried at 37 0c for 24 hour. Then teeth were equally divided into three experimental groups and one control group of 20 each. In group I teeth were instrumented with the hand stainless steel k files in group 2 instrumentation with rotary protaper universal in group 3-instrumentation with rotary protaper next and in group 4-No instrumentation is carried out.Then bacteria extruded were collected ,incubated in brain heart infusion broth for 24 hour at 37 0c and the colony forming units were counted.The result showed that all hand and rotary instrumentation technique extruded debris.Among all the instrumentation technique hand file extruded more number of debris and protaper next least number of bacteria.