Aim Recent concerns about the possible adverse effects of agricultural chemicals on health and environment have generated a considerable interest in biological alternatives. This study aimed to test the insecticidal potential of fungus Aspergillus flavus and revealed its genotoxic and cytotoxic effects using Spodoptera litura (Fabricius) as a model. Methods and Results The fungus was isolated from the surface of the dead insect and investigated for its insecticidal potential against S. litura by bioassay studies. Significant increase in mortality, prolonged development period and reduced adult emergence in S. litura were observed in larva fed on diet supplemented with fungal extract. In addition, fungus was also found to cause oxidative stress, DNA damage and cell death. Significantly higher percentages of necrotic cells and DNA damage were observed in larvae treated with fungal extract. Furthermore, DNA repair studies predicted the longevity of toxic effects induced by fungus. Phytochemical and ultra-high performance liquid chromatography studies revealed the presence of phenolic compounds in the extract and liquid chromatography-mass spectrometry indicated it to be a non-aflatoxin strain of A. flavus. Fungal extract was less toxic to mammalian cell lines as compared to cytotoxic drug doxorubicin (DOX) in the MTT assay. Conclusion The study highlights the insecticidal potential of A. flavus by revealing its genotoxicity and cytotoxicity causing potential. This is the first report showing the genotoxic and cytotoxic effects of the fungus A. flavus on S. litura. Significance and Impact of the Study: The study provides a useful insight to explore microbial agents as biopesticides in order to reduce various environmental as well as human health problems due to synthetic pesticides.