ABSTRACT Microbial diversity is strongly affected by the bottom-up effects of resource availability. However, because resource pools often exist as heterogeneous mixtures of distinct molecules, resource heterogeneity may also affect community diversity. To test this hypothesis, we surveyed bacterial communities in lakes that varied in resource concentration. In addition, we characterized resource heterogeneity in these lakes using an ecosystem metabolomics approach. Overall, resource concentration and resource heterogeneity affected bacterial resource-diversity relationships. We found strong relationships between bacterial alpha-diversity (richness and evenness) and resource concentration and richness, but richness and evenness responded in different ways. Likewise, we found associations between the composition of the bacterial community and both resource concentration and composition, but the relationship with resource composition was stronger. Last, in the surveyed communities the presence of resource generalists may have reduced the effect of resource heterogeneity on community composition. These results have implications for understanding the interactions between bacteria and organic matter and suggest that changes in organic matter composition may alter the structure and function of bacterial communities.