Nowadays, finding a safe control program is the aim of all researchers. The goal of this work is to investigate the effect of gamma radiation on the Entomopathogenic nematodes, Steinernema scapterisci and Heterorhabditis bacteriophora Poinar (HP88) efficacy were tested against larvae of cotton leaf worm, Spodoptera littoralis (Boisd.) under laboratory conditions. Results showed that 2 Gy irradiated S. scapterisci and H. bacteriophora were substantially effective in controlling S. littoralis larvae, while H. bacteriophora was more potent in controlling insect larvae. The results revealed that total protein concentration was significantly decreased (P < 0.05) after treatment with normal or irradiated H. bacteriophora or S. scapterisci. In addition, larvae infected with normal S. scapterisci or H. bacteriophora showed a significant elevation in phenoloxidase activity and represented significant reduce after treatment with 2 Gy irradiated S. scapterisci or H. bacteriophora as compared to control group. Also, lysozyme activity was significantly decreased after treatment with irradiated H. bacteriophora , but there was no significance with irradiated S. scapterisci , when compared with control. LDH activity was significantly high (p<0.05) in the haemolymph of larvae treated with normal or irradiated H. bacteriophora or S. scapterisci , as compared to control group. Furthermore among all treatments, 2 Gy irradiated H. bacteriophora was the most potent and efficient in the biomarkers changes. Therefore, it could be concluded that 2 Gy irradiated S. scapterisci and H. bacteriophora can serve within an integrated pest management (IPM) program in an agroecosystem.