Plants contain a wide range of potential phytochemicals that are target-specific, and less toxic to human health. The present study aims to investigate the metabolomic profile of Nephrolepis exaltata (L.) Schott and its potential for mosquito control by targeting Glutathione-S-Transferase, focusing on the larvicidal activity against Culex pipiens. Crude extracts (CEs) were prepared using ethanol, ethyl acetate and n-hexane. CEs have been used for assessment of mosquitocidal bioassay. The metabolomic analyses for CEs were characterized for each CE by gas chromatography–mass spectrometry (GC–MS). The most efficient CE with the highest larval mortality and the least LC50 was the hexane CE. Then, alkaline phosphatase (ALP) activity, and glutathione-S-transferase (GST) activity were assessed in larvae treated with the hexane CE. The results demonstrated a decline in protein content, induction of ALP activity, and reduction in GST activity. Finally, molecular docking and dynamic simulation techniques were employed to evaluate the interaction between the hexane phytochemicals and the GST protein. D-(+)-Glucuronic acid, 3TMS derivative and Sebacic acid, 2TMS derivative showed best binding affinities to GST protein pointing to their interference with the enzyme detoxification functions, potentially leading to reduced ability to metabolize insecticides.