We experimentally assessed kinetics and thermodynamics of electron transfer (ET) from the donor substrate (acetate) to the anode for a mixed-culture biofilm anode. We interpreted the results with a modified biofilm-conduction model consisting of three ET steps: (1) intracellular ET, (2) electron-hopping extracellular ET (EET), and (3) conductive EET governed by Ohm’s law. The steady-state current density was 0.82 ± 0.03 A/m2 in a miniature microbial electrolysis cell operated at fixed anode potential of -0.15 V versus standard hydrogen electrode. Illumina 16S-rDNA and -rRNA sequences showed that the Geobacter genus was less than 30% of the community of the biofilm anode. Although Monod kinetics for utilization of acetate were relatively slow, biofilm conductivity was high at 2.44 ± 0.42 mS/cm, indicating that the maximum current density could be as high as 268 A/m2 if only the conductive EET was limiting. Due to high biofilm conductivity, the maximum energy loss for the EET was negligible at 340 µV; the energy loss in the second ET step was only 20 mV, placing at least 87% of the energy loss at the intracellular step. The potential for a rate-limiting extracellular cofactor (EC) involved in the second ET was -0.15 V, which means that >99% of the EC was in the oxidized state, which reinforces that the intracellular ET was the main kinetic and thermodynamic bottleneck to electron transfer from donor substrate to the anode for a highly conductive biofilm.