A new family of PtFe catalysts supported on hierarchical porous carbon (HPC), with different porous sizes, was developed and tested as cathodes in oxygen reduction reaction in acetate-fed microbial fuel cells. The results obtained were compared with carbon black (CB), CB-PtFe supported catalysts. HPC400 was characterized by cyclic voltammetry, showing a good electrolyte accessibility in contrast to CB. The XPS analysis of HPC supports show a low content of oxygenated species on carbon surfaces. The morphological and structural properties of catalysts were characterized by SEM-EDX and XRD. The electrochemical performance was examined by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The CO stripping voltammetry was applied for the determination of the surface area of PtFe catalysts; these experiments were carried out in a three-electrode system. In oxygen reduction experiments by LSV, HPC400-PtFe and CB-PtFe show good current densities of − 10.3 mA cm−2 and − 9.09 mA cm−2, respectively. HPC300-PtFe presents the poorest performance (− 2.3 mA cm−2). The HPC500-PtFe is the catalyst with the major current density (− 26.19 mA cm−2). A similar behavior is obtained in microbial fuel cell (MFC) catalyst performances; the MFCs were evaluated during 16 days until biofilm formation and stabilization. An increment of the power density was observed with the number of days. The power density obtained for HPC500-PtFe was 3218.89 mW m−2, showing better performances compared with CB-PtFe (743.18 mW m−2), HPC400-PtFe (578.82 mW m−2), and HPC300-PtFe (266.82 mW m−2). Hierarchical porous structure in combination with large-sized macropores enhanced the catalytic activity of PtFe toward oxygen reduction reaction in microbial fuel cells.