Examining the impact of abiotic factors on the fatty acid profile of freshwater organisms is an important step in predicting the potential effects of climate change. I designed and deployed a heating system to manipulate the water temperature of a large mesocosm (>300,000 L) within a pond to simulate future predictions. Heating was observed near the piping system but not throughout the water column. In a separate experiment, the seasonal and depth-related variance in biomass and polyunsaturated fatty acids (PUFA) content of aquatic biofilm, grown on artificial substrates (unglazed ceramic tiles) was examined. Although summer biofilm typically had higher biomass and higher PUFA yield, fall biofilm typically contained higher PUFA content (standardized for biomass). Similarly, deeper biofilm accumulated higher organic matter but contained lower standardized PUFA content. These results suggest that abiotic factors may play a key role in determining the availability of nutritionally-important fatty acids in freshwater ecosystems.