Background Wastewater reuse represents a promising alternative source of water supply considering the water scarcity related to climate change. However, if not adequately treated, wastewater represents a source of microbiological health risk. The purpose of this work was to investigate the role of wastewater treatment on microbiological contamination by evaluating the possible risks associated with wastewater effluent reuse, taking into account new EU legislation (2020/741) on minimum requirements for water reuse. E. coli that produce Shiga toxins (STEC) and thermotolerant Campylobacter were monitored using an enrichment step associated with specific PCR, while Salmonella spp. and Legionella were detected with both cultural and molecular methods (PCR and q-PCR, respectively). Culture method was also used for the enumeration of different microbial indicators. The bacteria detection was compared in different wastewater plants with membrane bioreactor (MBR) system or with disinfection step with chlorine dioxide (ClO2). Moreover a comparison between molecular and culture methods was discussed. Results The results obtained showed good abatement performance for WWTPs equipped with MBR. The high concentrations of E. coli (range between 0.88 and 5.21 Log MPN/100 mL) and contamination by Salmonella spp. in effluent disinfected with ClO2 (17% of samples) showed the need to control the quality of this effluent. In addition, despite the absence of Legionella spp. with the culture method required by EU regulation, high concentrations of Legionella spp. (range between 2 and 7 log GU/L) and the presence of Leg. pneumophila with qPCR (15% of samples) highlight the need to carry out further investigations for reuse associated with aerosol formation (e.g. spray irrigation in agriculture). Conclusions The results obtained underline that the MBR technology can be suitable for wastewater reuse applications allowing to achieve the requirement proposed by the new European legislation. More attention should be given to wastewater reuse of effluents treated with ClO2. The use of the molecular methods for pathogens detection in wastewater could allow a more precautionary risks estimation associated with reuse. The overall results highlight that an evaluation of the effectiveness of the wastewater treatments is required for the prevention of a possible risk to public health.