Mosquitoes play a pivotal role in transmitting various disease agents that pose significant threats to public health. The extensive use of chemical pesticides to control mosquitos has resulted in physiological vector resistance besides negative environmental consequences. Phytochemicals represent bio-safe and eco-friendly alternatives for controlling disease vectors. This study aimed to assess the toxicity of a neem oil formulation (safe oil) against Culex pipiens (Diptera: Culicidae) third instar larvae. Mortality was recorded 24 hours post-treatment with four different concentrations of safe oil (0.028, 0.125, 0.25, 0.5 ppm). A significant increase in larval mortality was observed with rising concentrations, reaching a median lethal concentration (LC₅₀) of 0.112 ppm. In addition, the study investigated the biochemical impact of safe oil on total protein levels and the activities of detoxifying enzymes, acetylcholinesterase, and glutathione S-transferase in Cx. pipiens 3^rd instar larvae, 24 hours post-treatment with the LC₅₀ concentration. Treated larvae exhibited significantly reduced levels and activities across all assessed parameters compared to untreated controls. Histopathological examination of the larval midgut region treated with LC₅₀ revealed deformities in epithelial cells, detachment from the basement membrane, and disintegration of nuclei, forming irregular blebs protruding into the lumen. GC-MS analysis of the oil revealed the presence of 38 components with varying concentrations (0.18-8.23%). The neem oil formulation, specifically the chemical constituents present in it, was docked into the active pockets of GST and AChE receptors, providing additional support for the reported activities. These findings imply that safe oil and its components have a promising effect as larvicides for mosquito vector control. Additionally, this study is considered the first study that revealed the molecular docking of one of the neem oil formulations (safe oil).