The study explores the efficacy of Rosmarinus officinalis essential oil (EO) as a plant based insecticide and its effect on acetylcholinesterase (AChE) and antioxidant enzyme system in Sitophilus oryzae and Oryzaephilus surinamensis. GC–MS result revealed 2-methoxy-3-(2-propenyl)-phenol, 1,8-cineole, and camphor as the major component of the EO. As a fumigant, at 0.15 μl/ml EO exhibited 100% toxicity and antifeedant activity against the test insect pests without affecting seed germination. EO inhibited acetylcholinesterase enzyme activity (>40%) in both test insect pest. A significant impairment in the antioxidant enzyme system; superoxide dismutase (SOD), and catalase (CAT), along with the decrease in reduced glutathione (GSH) level was observed. However, in the case of O. surinamensis GSH level showed a biphasic response with an initial increase followed by a decline in its level. The findings revealed that the toxicity of EO might be associated with inhibition of AChE activity and oxidative imbalance. In view of the strong fumigant toxicity and antifeedant activity, test EO may be recommended as an eco-friendly alternative to synthetic insecticides.