Wastewater-fed microbial fuel cells (MFCs) are a promising technology to treat low-organic carbon wastewater and recover part of the chemical energy in wastewater as electrical power. However, the interactions between electrochemically active and fermentative microorganisms cannot be easily studied in wastewater-fed MFCs because of their complex microbial communities. Defined co-culture MFCs provide a detailed understanding of such interactions. In this study, we characterize the extracellular metabolites in laboratory-scale membrane-less MFCs inoculated with Geobacter sulfurreducens and Escherichia coli co-culture and compare them with pure culture MFCs. G. sulfurreducens MFCs are sparged to maintain anaerobic conditions, while co-culture MFCs rely on E. coli for oxygen removal. G. sulfurreducens MFCs have a power output of 128 mW m−2, compared to 63 mW m−2 from the co-culture MFCs. Analysis of metabolites shows that succinate production in co-culture MFCs decreases current production by G. sulfurreducens and that the removal of succinate is responsible for the increased current density in the late co-culture MFCs. Interestingly, pH adjustment is not required for co-culture MFCs but a base addition is necessary for E. coli MFCs and cultures in vials. Our results show that defined co-culture MFCs provide clear insights into metabolic interactions among bacteria while maintaining a low operational complexity.