Efficacy of spinosad and malathion loaded in eco-friendly biodegradable formulations was evaluated for controlling Culex pipiens larvae. Malathion (organophosphorus larvicide) and spinosad (naturally derived insecticide) were loaded on chitosan/alginate/gelatin capsules. Capsules were characterized by size measurement, scanning electron microscopy, Fourier transform infrared spectroscopy, and water uptake. In vitro release kinetics of the larvicides was studied in the running and stagnant water. Biochemical studies on the larvae treated with technical and formulated insecticides were also demonstrated. The results indicated that the released spinosad was active for a long time up to 48 and 211 days in the running and stagnant water, respectively. However, the capsules loaded with malathion showed larvicidal activity for 20 and 27 days in the running and stagnant water, respectively. Technical and formulated malathion and spinosad had an inhibition effect on acetylcholinesterase, carboxylesterase, and glutathione S-transferase. The results proved that the prepared capsules consisting of biodegradable polymers containing larvicides could be effective as controlled-release formulation against C. pipiens larvae for a long period.