This study explored acute impact of sulfamethoxazole (SMX) on mixed microbial culture by means of respirometric analysis and modeling of process kinetics. The culture was developed in a laboratory-scale activated sludge reactor fed with peptone mixture. The reactor was maintained at steady state at SRT of 10 days allowing the culture to sustain both heterotrophic and autotrophic fractions. Response of biomass under pulse feeding of 50 mg/L and 200 mg/L SMX was determined by monitoring corresponding oxygen uptake rate (OUR) profiles. Model calibration of OUR profiles enabled numerical assessment of the repressive action of SMX on all processes, heterotrophic growth; substrate binding and storage; two-step nitrification; endogenous respiration. The maximum growth rate of Nitrite Oxidizing Bacteria (NOB) was reduced by 35 %, compared to a slight decrease for Ammonia Oxidizing Bacteria (AOB). Upon exposure, significant substrate binding of around 40-50 % by SMX was observed, drastically reducing the amount of available carbon source for microbial growth. Substrate was directed towards storage with a 15-25% increase in the maximum storage rate. Pulse exposure to 200 mg/L of SMX induced a 50 % increase in the endogenous respiration rate, presumably to meet a higher need for maintenance energy potential for resistance and survival.