Biodiesels (fatty acid methyl esters) derived from oleaginous microbes (microalgae, yeast, and bacteria) are being actively pursued as potential renewable substitutes for petroleum diesel. Here, we report the engine performance characteristics of biodiesel produced from a microalgae (Chaetoceros gracilis), a yeast (Cryptococcus curvatus), and a bacteria (Rhodococcus opacus) in a two-cylinder diesel engine outfitted with an eddy current brake dynamometer, comparing the fuel performance to petroleum diesel (#2) and commercial biodiesel from soybeans. Key physical and chemical properties, including heating value, viscosity, density, and cetane index, for each of the microbial-derived biofuels were found to compare favorably to those of soybean biodiesel. Likewise, the horsepower, torque, and brake specific fuel consumption across a range of engine speeds also compared favorably to values determined for soybean biodiesel. Analysis of exhaust emissions (hydrocarbon, CO, CO2, O-2, and NOx) revealed that all biofuels produced significantly less CO and hydrocarbon than petroleum diesel. Surprisingly, microalgae biodiesel was found to have the lowest NOx output, even lower than petroleum diesel. The results are discussed in the context of the fatty acid composition of the fuels and the technical viability of microbial biofuels as replacements for petroleum diesel.