Mosquitoes are a critical global public health concern, causing millions of deaths each year due to the viral infections and diseases they spread. In recent decades, synthetic insecticides and repellents have been used to control mosquito populations and virus transmission, but they often harm the environment and non-target organisms, including humans. This research investigates the isolation, characterization, and structural elucidation of cis-13-Octadecenoic acid derived from Andrographis paniculata, and its effectiveness against the immature stages of Aedes aegypti and Culex quinquefasciatus. The extracts from A. paniculata underwent fractionation, with the most bioactive fractions being further purified and analyzed. Spectroscopic techniques were employed to confirm the structure of cis-13-Octadecenoic acid. Biological assays were then performed to evaluate its ovicidal, larvicidal, and pupicidal activities. cis-13-Octadecenoic acid exhibited notable ovicidal activity, causing 25 % mortality in Ae. aegypti eggs and 29 % mortality in Cx. quinquefasciatus eggs at a concentration of 2 ppm. Additionally, it showed strong larvicidal and pupicidal activities, with LC50 values of 2.67 ppm and 3.39 ppm for Ae. aegypti larvae and pupae, and 3.10 ppm and 2.93 ppm for Cx. quinquefasciatus larvae and pupae, respectively. These findings demonstrate the potential of cis-13-Octadecenoic acid as an effective natural compound for mosquito control, highlighting the broader importance of natural products as alternatives to synthetic insecticides in the management of vector-borne diseases.