Biodegradation of pharmaceuticals active compounds (PACs) in secondary effluents by using B. subtilis 2012WTNC as a function of beta-lactamase was optimized using response surface methodology (RSM) designed by central composite design (CCD). Four factors including initial concentration of bacteria (1-6 log(10) CFU mL(-1)), incubation period (1-14 days), incubation temperature (20-40 degrees C) and initial concentration of PACs (1-5 mg L-1) were investigated. The optimal operating factors for biodegradation process determined using response surface methodology (RSM) was recorded with 5.57 log(10) CFU mL(-1) of B. subtilis, for 10.38 days, at 36.62 degrees C and with 4.14 mg L-1 of (cephalexin/amoxicillin) with R-2 coefficient of 0.99. The biodegradation was 83.81 and 93.94% respectively. The relationship among the independent variables was significant (p < 0.05) with 95% of confidence level at the best operating parameters. The bioassay for PACs after the degradation process revealed that no residual antibiotic activity was detected of amoxicillin and cephalexin against E. coli and S. aureus after degradation using B. subtilis which reflects the higher potential of bacteria to biodegrade PACs in secondary effluents. B. subtilis has the potential for biodegradation of PACs in the secondary effluents.