Electron transfer (ET) behavior of bacteria varies significantly in a bio-electrocatalyzed environment. However, the exact mechanisms of ET towards electrodes are not well defined in most electrochemically active microorganisms. The bacterial cell structure and composition affects the ET properties as well as their growth. In the present study, disparity in ET between Gram positive (GPB, Bacillus subtilis) and Gram negative (GNB, Pseudomonas otitidis) bacteria (both differ in chemical and physical properties of cell wall/structure) and combination of both (GPB + GNB) was evaluated individually in bio-electrochemical cells (BEC_B, BEC_P and BEC_P+B). P. otitidis being a GNB exhibited mediated electron transfer (MET) through the redox shuttles detected as a peak in derivative of CV (DCV) analysis with an extra cellular electron transfer (EET) site potential of −36 mV corresponding to the phenazine derivative. GPB, B. subtilis exhibited direct electron transfer (DET) through the membrane bound proteins with peak potentials of 0.04 V, 0.211 V and 0.423 V that correspond to cytochrome-C, bc1 and aa3. Electron transfer capabilities in terms of electron transfer rate (K_app; 81 s⁻¹), redox catalytic currents (OC: 40 mA; RC: −50 mA), power density (63.3 mW m⁻²), sustainable anodic resistance (5 kΩ) and currents (5 mA) were found to be higher in GNB in comparison to GPB. The thin and permeable nature of the cell wall might have permitted the easy shuttling of redox mediators (MET) aiding efficient electron transfer in BEC_P in comparison to BEC_P+B and BEC_B attributed to the significant role of GNB as electrochemically active bacteria.