The aggregation of syntrophic Geobacter metallireducens and Geobacter sulfurreducens is beneficial for enhancing direct interspecies electron transfer (DIET). Although DIET was suspected to occur on the microbial community surface, the surface chemical speciation of such cocultured communities remains unclear. In order to better understand surface interactions related to DIET, the authors characterized a series of samples associated with syntrophic G. metallireducens and G. sulfurreducens using surface sensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis was used in spectral analysis. Our results show that the syntrophic Geobacter aggregates are significantly different from their planktonic cells, indicating a distinct chemical composition (i.e., amino acids, fatty acids, and lipids) and structure formed on their surface. Among these characteristic components, amino acid fragments dominated in the variance, suggesting the importance of proteins in the coculture. Additionally, the quorum sensing signal molecule N-butyryl-l-homoserine lactone was observed in cocultured Geobacter aggregates, implying its role in syntrophic growth and aggregate formation. Furthermore, the electron acceptor organism G. sulfurreducens was shown to be the dominant species in syntrophic communities that drove the syntrophic growth. These results demonstrate that unique chemical compositions distinguish syntrophic Geobacter aggregates from planktonic cells and suggest that ToF-SIMS may be a promising tool to understand the syntrophic mechanism and investigate interspecies electron transfer pathways in complex biofilms.