Electroactive biofilms (EABs) generated from mixed inocula are attractive due to their unique direct extracellular electron transfer abilities and potential use in water pollution control. In this study, for the first time, we identified a chemical that can be used for EAB regulation (both inhibition and promotion). We confirmed that tobramycin, an antibiotic previously demonstrated to inhibit the activity of EABs, is an agonist of EAB formation at subminimal inhibitory concentrations (sub-MICs). Compared to the control, at tobramycin concentrations of 0.05 (1/80 MIC) and 0.1 mg/L (1/40 MIC), the time required to reach 3 A/m was shorter, and the limiting current densities increased by 17%. The enhanced EAB activity was primarily attributed to the 50% increase in biomass density from 289 ± 21 to 434 ± 12 μg protein/cm and the increased biofilm thickness from 28 ± 1 to 37 ± 0.5 μm. Geobacter species in the microbial communities were selectively increased from 76% to 82%, and their abundance was estimated to increase by 1.63-fold. The accelerated growth was further confirmed using the model strain G. sulfurreducens PCA. Transcriptomic analysis revealed that 0.05 mg/L of tobramycin led to a significant upregulation of genes related to cytochromes and the type IV pilus, suggesting a possible mechanism for the observed current enhancement. These findings extend our knowledge of the regulation of EAB formation by antibiotics and the selective enrichment of Geobacter from a mixed culture, with broader implications on the potential impact of trace antibiotics on the dissimilatory metal reduction process in water environment.