Background and Objective: Antibiotic resistance (ABR) is of primary concern for Public Health. As reported by the European One Health Action Plan against Antimicrobial Resistance, there is the need to monitor the presence of antibiotics, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment, especially in wastewater. Moreover, the global action plan on antimicrobial resistance underlines the need to obtain strong evidence associated to the role of wastewater and environment as contributors to the development and spread of ABR. The aim of this study is to evaluate the ABR spreading in different treatment steps of wastewater treatment plant (WWTP) also considering the possible reuse of effluent in agriculture. Methods: Samples collected at different steps of a WWTP developed for reuse were investigated for the presence of antibiotics (ampicillin, tetracycline, sulfamethoxazole), heavy metals, the antibiotic resistance rate of ARB (ampicillin, tetracycline, and sulfamethoxazole resistant bacteria) and ARGs (blaTEM, tetA, sulII). Results: The Results obtained highlight the presence of ARB and ARGs for the antibiotics tested in WWTP, with a decreasing trend during the wastewater treatment, as also observed for some antibiotics and heavy metals. Although there is generally no significant difference in the ABR rate, a higher average value of this rate is observed during the wastewater production, suggesting a possible positive selective pressure of ARB by some steps of treatment. A correlation was also observed between the presence of some heavy metals and ABR rate. Conclusions: The results obtained suggest the need to carefully monitor the role of WWTPs in the spreading of antibiotic resistance, also considering the One Health approach. Furthermore, the results obtained could suggest interventions to reduce the human health risk associated with wastewater reuse in agriculture.