In the quest for innovative alternatives for metabolizable energy, the focus has shifted to the biomass of insect larvae. This study aimed to extract oil from the larval biomass of the housefly (Musca domestica L. 1758, Diptera: Muscidae) and to analyze the oil yield, fatty acid profile, quality indices, infrared spectra, metal concentrations, antioxidant activity, and cytotoxicity using a mouse fibroblast cell line (L929). The results demonstrated that the oil yield extracted from housefly larvae biomass was 26.3 %. The fatty acid profile revealed a high content of monounsaturated fatty acids (50.6 %), polyunsaturated fatty acids (43.9 %), and saturated fatty acids (5.5 %), with oleic acid and linoleic acid as the predominant components. The peroxide index, acidity, and saponification value were measured at 4.30 meq/Kg, 0.021 mg KOH/g, and 0.65 mg KOH/g, respectively, indicating excellent oil quality. Infrared analysis revealed similar functional groups in both the larval biomass and the extracted oil; however, the O–H and C-O functional groups were absent in the oil. The housefly larvae biomass contained essential minerals, including Cu, Fe, K, Na, and Zn. The extracted oil showed low concentrations of potentially toxic elements such as Cr and Ni. Importantly, housefly oil exhibited no cytotoxic effects on healthy cells while reducing reactive oxygen species (ROS) levels and enhancing the activities of superoxide dismutase (SOD) and catalase (CAT) enzymes, highlighting its significant antioxidant potential. Given their chemical compositions, both the housefly larval biomass and oil show promise for applications in human and animal nutrition.