Abstract Three different generations (Gen-I, -II and -III) of microbial fuel cell (MFC), distinguished by their historical development and mechanisms of electron transfer, were compared. Gen-I utilised synthetic redox mediators combined with Escherichia coli . In contrast, the Gen-II exemplar utilised the natural mediating properties of sulphate/sulphide with the sulphate reducing species Desulfovibrio desulfuricans . Gen-III MFCs were based on the anodophillic species Geobacter sulfurreducens and required no soluble mediator. Each type of MFC was operated under similar environmental conditions. In terms of substrate to power conversion efficiency, Gen-II was most efficient (64.52%), followed by Gen-III (47.38%) and Gen-I (28.12%). When output was expressed as power/unit of cells, Gen-III was 28-fold higher by comparison (33.72 × 10 15 e/μg cells). For comparative purposes, these results were produced using equal rather than optimal circuit loads. Under optimal loading conditions, Gen-III produced on average five-fold higher power than under equal load and the conversion efficiency was 95%. To the best of the authors’ knowledge, this is the first time that these three types of MFC have been experimentally compared under similar conditions. Gen-II and -III but not Gen-I may be used advantageously in wastewater treatment and power generation from organic matter.