This study investigated the bioelectrical performance of a single-chamber microbial fuel cell (SCMFC) fueled with acetate as the electron donor and inoculated with municipal solid waste rejected fractions (MSWRFs) as a microbial inoculum. The molecular characterization of the bacterial community structures of the anodic biofilm was conducted based on 16s RNA gene sequencing. The results indicated that the highest open-circuit voltage (OCV) was 797 mV and the system had a maximum power density of 134.5 mW/m² at a stable current density of 328 mA/m². The microbial fuel cell’s (MFC) columbic efficiency (CE) was 55% at a maximum substrate degradation rate of about 86.6% based on COD removal efficiency. The molecular analysis of the anodic bacterial isolates indicated that the phylogenetic bacterial mixture was dominated by seven strains with similarity percentage above 99% for each strain: <i>Enterococcus faecalis, Clostridium butyricum, Bacillus sp., Bacillus subterranous, Enterobacter celoaca,</i> <i>Klebsiella pneumonia,</i> and <i>Escherichia coli</i>. These results suggested that MSWRFs bacterial consortia have a moderate symbiotic structure as indicated by electrons release in parallel with substrate decomposition.