Pre-regulation coal mining and subsequent acid mine drainage (AMD) have drastically altered stream quality in the Appalachian region of the USA. Streams impaired by AMD often times demonstrate lowered pH, increases in specific conductance, and increase in dissolved metal concentrations. These changes in the chemical environment are reflected in the biotic community with drastic reductions in diversity and biomass. Recently, there has been an increase in applying traditional measures of food quality to understand how the biofilm community is altered by environmental condition and use for stream quality monitoring. The purpose of this study was two-fold: (1) to use fatty acid profiles to distinguish between biofilm communities in AMD impaired and unimpaired streams and (2) to determine the consistency of biofilm fatty acid profiles throughout the summer sampling period. Impaired streams showed significantly lower pH and increased specific conductance. Biofilm samples from the AMD impaired streams had lower fatty acid content with a decreased proportion of polyunsaturated fatty acids. Fatty acid profiles easily and rapidly separated biofilm communities into their respective categories, either as being impaired by AMD or unimpaired by AMD, using multivariate statistical approaches. Fatty acid profiles were similar within stream type throughout the summer sampling season, and the profiles were correlated to pH and specific conductance. The results of this study suggest that fatty acid profiles can rapidly and accurately categorize the biofilm community responses to environmental impairment.