This study investigates the performance of acetate feed membrane less single chamber microbial fuel cell and physical characterization of the bio film present on the anode surface using Scanning Electron Microscope (SEM) and 16S rRNA analyzer. The performance has been investigated using Teflon treated carbon paper with 0.3 mg/cm2 Pt/C loaded as a cathode and carbon paper as an anode. The maximum open circuit potential is noticed as 791 mV, the system successfully revealed a maximum power density of 86.1 mW m-2 at stable current density of 354 mA m-2 with high coulombic efficiency of 65% at maximum degradation rate of 96%. SEM showed the dense adherence of microorganisms on the anode. 16S rRNA sequencing results indicates phylogenetic mixture in the communities of anodic biofilm and there is no single dominant bacterial species. The dominant phyla are Firmicutes, Gamma Proteobacteria, Alpha Proteobacteria, Actinobacteria, with ten dominant microbial strains: Bacillus firmus, Shewanella profunda, Bacillus isronensis, Brevundimonas bullata, Pseudomonas putida, Planococcus citreus, Micrococcus endophyticus, Acinetobacter tandoii, Bacillus safensis and Shewanella xiamenensis.