Abstract Microbial fuel cells (MFCs) rely on microbial conversion of organic substrates to electricity. The optimal performance depends on the establishment of a microbial community rich in electrogenic bacteria. Usually this microbial community is established from inoculation of the MFC anode chamber with naturally occurring mixed inocula. In this study, the electrochemical performance of MFCs and microbial community evolution were evaluated for three inocula including domestic wastewater (DW), lake sediment (LS) and biogas sludge (BS) with varying substrate loading ( L sub ) and external resistance ( R ext ) on the MFC. The electrogenic bacterium Geobacter sulfurreducens was identified in all inocula and its abundance during MFC operation was positively linked to the MFC performance. The LS inoculated MFCs showed highest abundance (18% ± 1%) of G. sulfurreducens , maximum current density [ I max = (690 ± 30) mA·m − 2 ] and coulombic efficiency (CE = 29% ± 1%) with acetate as the substrate. I max and CE increased to (1780 ± 30) mA·m − 2 and 58% ± 1%, respectively, after decreasing the R ext from 1000 Ω to 200 Ω, which also correlated to a higher abundance of G. sulfurreducens (21% ± 0.7%) on the MFC anodic biofilm. The data obtained contribute to understanding the microbial community response to L sub and R ext for optimizing electricity generation in MFCs.