17-α-ethynylestradiol (EE2) biodegradation was conducted using biofilm reactor in a medium containing both EE2 and glucose as organic carbon sources and in a medium without glucose and EE2 as the sole organic carbon source. The factors affecting the biodegradation are initial pH, initial EE2 concentration, initial nitrogen content, and biomass volume. Response surface methodology (RSM) and central composite design (CCD) were applied to determine the optimum operating conditions with the % degradation and the degradation efficiency as responses. The ANOVA revealed that EE2 concentration is the most significant factor that influences the % degradation and the biomass volume is the most significant factor that influences the degradation efficiency. Verification runs at optimum conditions showed that there were some peaks that appeared besides the peak of EE2 indicating that EE2 was biochemically transformed. The Monod kinetic and first order suit well for the EE2 biodegradation.