Background: Mosquito is a big threat to the human health. Mosquito-borne diseases cause millions of death to the human beings. Hence, a permanent solution is eagerly to be established to control its excessive growth in stagnant water. Portulaca oleracea Linn is a natural larvicidal agent, which contains active ingredients such as linolenic acid, linoleic acid (omega-3 fatty acids). These bioactive compounds may be responsible for its larvicidal properties on mosquito. Objective: The present study is focussed on identifying the bioactive compounds, such as linolenic acid, through GC–MS, and analyzing the larvicidal efficacy of Portulaca oleracea L. against Culex quinquefasciatus and Anopheles stephensi larvae. Methods: Preliminary phytochemical analysis, total protein content, total carbohydrate content, total phenol content, total flavonoid content and GC–MS analysis exhibited the presence of rich phytoconstituents in Portulaca oleracea. DPPH analysis was carried out to analyze the antioxidant potential of plant extract. Larvicidal activity and histological change were detected to evaluate the efficacy of Portulaca oleracea L. on the Culex quinquefasciatus and Anopheles stephensi larvae. Results: Aqueous and ethanol leaf extracts of Portulaca oleracea L. against both Culex quinquefasciatus and Anopheles stephensi larvae, showed a very good larvicidal activity at 500 μg/mL among the various concentrations. Histological damages of mosquito larvae were observed when treated with the Portulaca oleracea L. extract, and provided further evidence for its larvicidal activity. Conclusion: This study concluded that the plant Portulaca oleracea L. contained many useful bioactive compounds, can be a strong larvicidal agent against both Culex quinquefasciatus and Anopheles stephensi larvae. The molecular mechanism for the larvicidal activity will be identified in future studies.