Antibiotics are drugs used on both animals and humans. However, when their residues or metabolites are released into the environment, it can cause microbial resistance in water bodies, including wastewater treatment plants. In this study, extended spectrum β-lactamases (ESBLs) were monitored in an anaerobic-aerobic combined biological treatment system. The enzymes Oxacillinase (OXA), Cefotaximases (CTX) and New Delhi metallo-β-lactamase (NDM-1) were added to the treatment and analyzed by qPCR (Quantitative Polymerase Chain Reaction) and plate count using a selective medium. Quantification of the effect of adding these enzymes was performed before, during and after they were added in order to establish the behavior of the treatment system under three different conditions. Anaerobic reactors proved more efficient at treating wastewater containing the β-lactamases OXA, CTX and NDM-1 than the ASP3 aerobic reactor that was working individually. However, an anaerobic-aerobic treatment train was much more effective at removing organic matter, even in the presence of β-lactamase.