In the present research work, a non-edible oil source Cucumis melo var. agrestis (wild melon) was systematically identified and studied for biodiesel production and its characterization. The extracted oil was 29.1% of total dry seed weight. The free fatty acid value of the oil was found to be 0.64%, and the single-step alkaline transesterification method was used for conversion of fatty acids into their respective methyl esters. The maximum conversion efficiency of fatty acids was obtained at 0.4 wt% NaOH (used as catalyst), 30% (methanol to oil, v/v) methanol amount, 60 ℃ reaction temperature, 600-rpm agitation rate and 60-min reaction time. Under these optimal conditions, the conversion efficiency of fatty acid was 92%. However, in the case of KOH as catalyst, the highest conversion (85%) of fatty acids was obtained at 40% methanol to oil ratio, 1.28 wt% KOH, 60 ℃ reaction temperature, 600-rpm agitation rate and 45 min of reaction time. Qualitatively, biodiesel was characterized through Fourier transform infrared spectroscopy (FTIR) and gas chromatography and mass spectroscopy (GC–MS). FTIR results demonstrated a strong peak at 1742 cm_-1, showing carbonyl groups (C=O) of methyl esters. However, GC–MS results showed the presence of twelve methyl esters comprised of lauric acid, myristic acid, palmitic acid, non-decanoic acid, hexadecanoic acid, octadecadienoic acid and octadecynoic acid. The fuel properties were found to fall within the range recommended by the international biodiesel standard, i.e., American Society of Testing Materials (ASTM): flash point of 91 ℃, density of 0.873 kg/L, viscosity of 5.35 cSt, pour point of -13 ℃, cloud point of -10 ℃, total acid number of 0.242 mg KOH/g and sulfur content of 0.0043 wt%. The present work concluded the potential of wild melon seed oil as excellent non-edible source of bioenergy.