The present study was conducted to isolate the causal agent of necrotic enteritis (NE) from broiler chickens. A total of 50 jejunal samples from necropsied broiler chickens were collected for isolation of Clostridium perfringens, the causal agent of NE. The novel method for isolation of Clostridium perfringens was stab culture in association with pouring of olive oil on the surface of the stab culture. The collected samples in nutrient broth were kept in a candle jar and incubated for 24 hrs at 37°C. For maintenance of anaerobic condition olive oil (2-3 cm) were poured on surface of culture broth in test tube. In Gram's staining, the morphology of the isolated bacteria was Gram-positive, rod-shaped, anaerobic, spore-forming single or paired in arrangement. The addition of olive oil on the surface of stab culture was effective for the growth of Clostridium perfringens. The characterization of Clostridium perfringens was then performed by other routine cultural study, staining procedure and different types of biochemical tests. The occurrence of necrotic enteritis was 8%.