Present dependence on fossil sources for meeting energy demand globally is unsustainable as a result of non‐renewable and depleting supplies. Therefore, this study focuses on production and characterization of biodiesel by Chlorococcum littorale (Chlorococcum sp.) isolated from fish pond in Owode fish farm, Nigeria. Biomass concentration, lipid extraction and quantification were determined by filtration, solvent extraction and gravimetric methods. Extracted lipid was transesterified to diesel through process catalyzed by concentrated tetraoxosulphate (VI) acid. The constituents of fats in biodiesel produced were examined through chromatography of gas and spectroscopy of mass. Model prediction was used to assess physical and chemical parameters of biodiesel. The data gathered was subjected to statistical analysis. Chlorococcum sp. had biomass concentration (4.42 ± 0.02 g/L), Lipid content (67.75% and biodiesel yield (96.33%). Biodiesel had 48.3%, 29.20%, and 22.50% fatty acids that are saturated, monounsaturated and polyunsaturated, respectively. The biodiesel produced has the kinematic viscosity (2.74 mm2/s at 40°C); point of flash (245°C); point of cloud (7°C); point of pour (−2°C) and density (0.8760 g/cm3). Furthermore, the acidity level (0.44 mgKOH/g), value of saponification (186.49 mgKOH/g), value of peroxide (3.25 meq/kg), iodine content (38.27 mgI2/g) as well as the free fatty acid (1.34%), number of cetane (65.78), oxidative resistance (7.33 h), saturated factor of long chain (28.16°C), plugging point of cold filter (35.62°C) and high heating value (43.24°C) fell within required standard by international body. Thus, Chlorococcum sp. is ideal microalgae isolate for the biodiesel production. The biodiesel produced conform satisfactorily with the acceptable global standard.