Detection and partial degradation of the cocaine metabolite benzoylecgonine in synthetic and real human urine is accomplished using single-chamber air-cathode microbial fuel cells. Microbial fuel cells generate voltage in the range of 0.2-0.26 V using synthetic urine or real human urine obtained from both cocaine users and drug-free individuals. Concentrations of benzoylecgonine up to 1000 ng/mL are treated in the fuel cells, and electricity generation is decreased with respect to increasing concentrations of benzoylecgonine. Power density, current density, chemical oxygen demand removal and total carbohydrate removal data confirm that, in comparison to the synthetic urine, fuel cell performance decreases using benzoylecgonine-containing human urine as the medium. In the fuel cells, benzoylecgonine levels decrease by 14% in 24 h of incubation, as determined by mass spectrometry results. According to the computational chemistry analysis, cation form 2 of the benzoylecgonine might limit transfer of electrons from the microorganisms to anode. In conclusion, microbial fuel cell technology is shown to exhibit a potential for use as biosensors for detection and quantification of cocaine metabolite benzoylecgonine in real human urine.