Indirect detection and quantification of the neomycin sulfate antibiotic was accomplished in microbial fuel cells. Performance of the microbial fuel cells was examined on the basis of the following parameters; voltage generation, power density, current density and coulombic efficiencies. Removal of neomycin sulfate was monitored using LC-MS/MS in parallel with chemical oxygen demand and total carbohydrate removal. While neomycin sulfate was partially degraded, microbial fuel cell performance appeared to be affected and eventually inhibited by neomycin sulfate on a concentration-based fashion. In order to further examine the neomycin sulfate biosensing activity of the microbial fuel cell, a computational chemistry approach was used to obtain the information about the highest occupied molecular orbital-lowest unoccupied molecular orbital energy values of outer electron orbitals, their distribution, and ionization potentials (IPs). The results showed that electroactive bio-film-based MFCs can be used for sensitive detection of neomycin sulfate found in wastewaters.