In this work, biodegradation of biodiesel samples were studied. Biodiesel is a renewable diesel fuel of domestic origin derived from a variety of fats and oils by a transesterification reaction. It consists of the alkyl esters, usually methyl esters, of the fatty acids. Biodiesel is one of the most interesting ways to produce renewable liquid fuel for transportation. Advantages of biodiesel include domestic origin, reducing the dependency on imported petroleum, biodegradability, high flash point, and inherent lubricity in the neat form. There are many methods for biodegradation. The biodegradation is quantified by trapping carbon dioxide (CO2) formed in the vessel during biodegradation. Among them, the CO2 evolution method is relatively simple, economical, and environmentally safe. Comparison of the biodegradability of biodiesel to conventional reference diesel fuel (2-D) was studied. The 2-D was used for a comparison petroleum diesel in all of the studies. The results demonstrate that all biodiesels assessed are biodegradable, with similar shaped curves with 80.4 to 91.2% biodegradation after 30 days, whereas the 2-D sample reached only 24.5% biodegradation. Biodegradation is degradation caused by biological activity, particularly by enzyme action leading to significant changes in the material's chemical structure. The biodegradability of biodiesel is dependent on the chemical structure of the fatty acids.