The heterotrophic utilization of organic substrates by diatoms is likely an important survival strategy when light levels are too low for photosynthesis. The objectives of this study were: (1) to determine if heterotrophic utilization of a large array of organic compounds by eight common freshwater benthic diatom taxa was light-dependent, and (2) to determine if organic substrate utilization patterns differed between dark-grown diatoms and bacteria as a possible means of reducing competition by niche separation. Eight light- and dark-grown diatom taxa and five bacterial species were incubated in 96-well Biolog® Microtiter plates with each well containing 1 of 95 different organic substrates. Oxidation rates of each organic substrate were measured through time. There was a substantial increase in the number of organic substrates oxidized by diatoms grown in the dark compared to their light-grown counterparts, indicating that the transport systems for these molecules may be light activated. Therefore, diatoms likely only utilize these metabolically expensive uptake mechanisms when they are necessary for survival, or when substrates are plentiful. A principal components analysis indicated discernible differences in the types of organic-C substrates utilized by dark-grown diatoms and bacteria. Although bacteria were able to oxidize a more diverse array of organic substrates including carboxylic acids and large polymers, diatoms appeared to more readily utilize the complex carbohydrates. By oxidizing different organic substrates than bacteria, heterotrophically metabolizing diatoms may be reducing direct competition and enhancing coexistence with bacteria.