The cotton leafworm, Spodoptera littoralis (Bosid.), is a major pest in African and Asian nations that attacks a wide variety of host plants. This study was conducted to assess the effectiveness of effective microorganisms (EMs) on the biological and physiological features of S. littoralis larvae. Five concentrations (100, 200, 300, 400, and 500 ppm) of EMs were tested. Antifeedant activity, food consumption index, the efficiency of converting digested food, the efficiency of converting ingested food, relative growth rate, and approximate body tissue of the fourth larval instar of S. littoralis were determined. Moreover, carbohydrate enzyme activities (amylase, trehalose, and invertase), total protein, and total lipids of S. littoralis larvae were measured to elucidate the mode of action of the tested agent in the S. littoralis’s larval stage. The EMs at 500 ppm had a substantial impact on antifeedant activity, nutritional indices, egg deposit reduction, and hatchability in S. littoralis during the five days. All concentrations interrupted S. littoralis’s life cycle and developmental phases. Furthermore, all concentrations were quite useful in lengthening the developmental stages of S. littoralis. In addition, Ems affected the biochemical activities of larvae, leading to disturbances in carbohydrate, lipid, and protein levels. From this study, EMs can be used as a bioinsecticide alternative to traditional insecticides against S. littoralis and may be compatible with integrated pest management approaches.