Bio-pesticides, derived from natural sources that target pest’s nervous systems directly, represent a sustainable alternative to chemical pesticides. The widespread use of chemical pesticides has led to profound ecological and health concerns, including biodiversity decline, genetic variability in plant populations, water pollution, and human health risks like cancer. This study focuses on extracting an environmentally friendly bio-pesticide from custard apple seed kernels, emphasizing high yield and efficacy. The extraction process employed advanced batch processing using Soxhlet extractor and rota-evaporator, ensuring efficient separation and purity of the bio-active compounds. Gas Chromatography (GC) was employed to meticulously characterize the chemical composition of the extracted samples. The effect of solvent, particle size and time of contact were determined. Furthermore, the bio-pesticide’s effectiveness against pests infesting soil and leaf surfaces was rigorously evaluated. The results of GC analysis revealed that Oleic acid and Palmitic acid are prominent bio-active components responsible for insect mortality. Furthermore, it is noteworthy that hexane extraction yielded the highest percentage (70%) of bio-pesticide. This research advances sustainable pest management strategies by developing natural bio-pesticides from agricultural by-products. Emphasizing eco-friendly solutions, the study aims to mitigate the environmental impact of conventional pesticides while promoting agricultural sustainability and ecosystem health.