Naturally occurring microorganisms in soil matrices play a significant role in overall hydrocarbon contaminant removal. Bacterial and fungal degradation processes are major contributors to aerobic remediation of surface contaminants. This study investigated degradation of conventional diesel, heating diesel fuel, synthetic diesel (Syntroleum), fish biodiesel and a 20% biodiesel/diesel blend by naturally present microbial communities in laboratory microcosms under favorable environmental conditions. Visible fungal remediation was observed with Syntroleum and fish biodiesel contaminated samples, which also showed the highest total hydrocarbon mineralization (>48%) during the first 28 days of the experiment. Heating diesel and conventional diesel fuels showed the lowest total hydrocarbon mineralization with 18–23% under favorable conditions. In concurrent experiments with growth of fungi suspended on a grid in the air space above a specific fuel with little or no soil, fungi were able to survive and grow solely on volatile hydrocarbon compounds as a carbon source. These setups involved negligible bacterial degradation for all five investigated fuel types. Fungal species able to grow on specific hydrocarbon substrates were identified as belonging to the genera of Giberella, Mortierella, Fusarium, Trichoderma, and Penicillium.