Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) is a major insect pest that severely affects various crops. Our study provides new insights by combining field efficacy trials with enzymatic analysis to evaluate the effects of emamectin benzoate mixtures with other insecticides (lufenuron, cypermethrin, chlorpyrifos, and spinosad) against S. littoralis. The aim of our work was to investigate the effectiveness of five insecticides, i.e., emamectin benzoate, lufenuron, cypermethrin, chlorpyrifos, and spinosad, for controlling this pest under field conditions during two consecutive seasons (2023–2024). Each insecticide was applied individually at the recommended rate, while some were mixed with emamectin benzoate at half its recommended rate. The results indicated that emamectin benzoate was the most effective insecticide, followed by lufenuron. The joint action of emamectin benzoate (LC25) and its mixtures with other insecticides (chlorpyrifos, spinosad, cypermethrin, and lufenuron) at various concentrations (LC50) against second- and fourth-instar S. littoralis larvae was evaluated. Results showed additive effects with chlorpyrifos, lufenuron, and cypermethrin, while potentiation occurred with cypermethrin (LC50) and chlorpyrifos (LC50). Antagonistic effects were observed in the combination of emamectin benzoate with spinosad (LC25 and LC50). This study concluded that applying insecticides individually is more cost-effective for managing cotton leafworm infestations in cotton crops. Additionally, enzyme activity analysis showed significant changes in alpha-esterase, beta-esterase, carboxylesterase, acetylcholinesterase, and glutathione S-transferase levels in larvae treated with different insecticide combinations.