Many studies attest to the pollution of surface water, wastewater and water intended for human consumption by organic molecules including emerging drug residues at trace levels. These emerging micropollutants are mostly little or not removed by sewage treatment plants and can consequently be found in the natural environment. The occurrence of antibiotic drug Sulfonamides in influent and effluent samples from three Tunisian Wastewater Treatment Plants was evaluated. The application of optimized multi-residue method for the simultaneous quantification and confirmation of 9 commonly used antimicrobials was carried out by on-line solid phase extraction liquid chromatography tandem mass spectrometry (SPE–LC–MS/MS). Isotopically labeled compounds Sulfadimethoxine d6 and Sulfamerazine 13C6 were used as surrogate internal standards to compensate for possible matrix effects. The presence of 9 antimicrobials is confirmed in the influent samples. Exclusively sulfamethoxazole was detected in both influent and effluent samples of three WWTPs (WWTP1, WWTP 2 and WWTP 3) at concentrations of (1.1–5.3µg/L), (0.3–0.6 µg/L) and (0.4–0.5 µg/L), respectively. Moreover, the highest concentration of sulfamethoxazole observed i.e. 5.3 μg/L in the effluent sample is mainly due to the great size of WWTP 1 and the large volumes of wastewater inflow which is in accordance to the largest population that it serves with 400.000 inhabitant equivalents.