The fuel qualities of several biodiesels containing highly saturated, mono, and poly unsaturated fatty acids, as well as their combustion and exhaust emission characteristics, were studied. Six biodiesel samples were divided into two groups based on their fatty acid composition, including group 1 (coconut, castor, and jatropha) and group II (palm, karanja, and waste cooking oil biodiesel). All fuels (in both groups) were tested in a single-cylinder off-road diesel engine. Castor and karanja biodiesel, both rich in mono-unsaturation level, have a high viscosity of about 14.5 and 5.04 mm2/s, respectively. The coconut and palm biodiesels are rich in saturation level with cetane numbers of 62 and 60, respectively. In both groups, highly saturated and poly-unsaturated methyl esters presented better combustion efficiency and less formation of polluted emissions than mono-unsaturation. At full load, coconut and palm biodiesel displayed 38% and 10% advanced start of combustion, respectively, which reduced ignition delay by approximately 10% and 3%, respectively. Mono-unsaturated methyl esters exhibited a higher cylinder pressure and heat release rate, which results in higher NOx gas emissions. The group II biodiesels showed about 10–15% lower exhaust emissions owing to an optimum level of fatty acid composition. Our study concluded that highly saturated and poly-unsaturated fatty acid performed better than mono-unsaturated biodiesels for off-road engine application.