Inoculum is critical for the start-up and performance of microbial fuel cells (MFCs). The effluent of mature MFCs is usually used as inoculum for the start-up of immature MFCs. However, the effluent of mature MFCs contains bacteria both from anode biofilm (ASB) and cathode biofilm (CSB). Here, ASB and CSB and their mixtures were used as inoculum in the start-up of MFCs in order to gain more insight into the influence of CSB on the start-up of MFCs. Compared to anode inoculum-enriched MFCs, using cathode inoculum reduced start-up time from 5 d to 3 d. The time needed for scavenging oxygen was reduced from 900 min to 600 min, maximum power density was 19% lower (691 mW/m² vs 823 mW/m²), and the charge transfer resistance increased from 29.0 Ω to 48.3 Ω. The decreased start-up time and power generation of cathode inoculum-enriched MFCs was attributed to the increasing abundance of Azospirillum (80.02% vs. 12.68%) and the decreasing abundance of Geobacter (9.08% vs. 61.25%). This research suggested that CSB in the effluent of mature MFCs, when used as inoculum, has a side-effect on the start-up of MFCs.