Abstract An electrochemically active bacteria Pseudomonas aeruginosa IIT BT SS1 was isolated from a dark fermentative spent media fed anode, and a bioaugmentation technique using the isolated strain was used to improve the start-up time of a microbial fuel cell (MFC). Higher volumetric current density and lower start-up time were observed with the augmented system MFC-PM (13.7 A/m3) when compared with mixed culture MFC-M (8.72 A/m3) during the initial phase. This enhanced performance in MFC-PM was possibly due to the improvement in electron transfer ability by the augmented strain. However, pure culture MFC-P showed maximum volumetric current density (17 A/m3) due to the inherent electrogenic properties of Pseudomonas sp. An electrochemical impedance spectroscopic (EIS) study, along with matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis, supported the influence of isolated species in improving the MFC performance. The present study indicates that the bioaugmentation strategy using the isolated Pseudomonas sp. can be effectively utilized to decrease the start-up time of MFC.