The study evaluated the inhibitory impact of antibiotics on the biodegradation of peptone mixture by an acclimated microbial culture under aerobic conditions. A fill and draw reactor fed with the peptone mixture defined in the ISO 8192 procedure and sustained at steady state at a sludge age of 10 days was used as the biomass pool with a well-defined culture history. Acute inhibition experiments involved running six parallel batch reactors seeded with biomass from the fill and draw reactor and the same peptone mixture together with pulse feeding of 50 mg/L and 200 mg/L of Sulfamethoxazole, Erythromycin and Tetracycline. Substrate utilization was evaluated by observing the respective oxygen uptake rate profiles and compared with a control reactor, which was started with no antibiotic addition. While all available external substrate was removed from solution, addition of antibiotics induced a significant decrease in the amount of oxygen consumed, indicating that a varying fraction of peptone mixture was blocked by the antibiotic and did not participate to the on-going microbial growth mechanism. This observation was also compatible with the concept of the uncompetitive inhibition mechanism, which defines a similar substrate blockage through formation of an enzyme-inhibitor complex.