A large number of tourist structures in Venice (Italy) have small sized on-site treatment systems for their wastewater. Due to its historical characteristics, the city has no public sewerage system and untreated hotel wastewater represents a serious hazard for its lagoon environment. This study focused on the wastewater facilities installed in two hotels adopting an Activated Sludge Sequencing Batch Reactor (AS-SBR) and an Ultra-Filtration Membrane Biological Reactor (UF-MBR). Their performance was checked in terms of both traditional physico-chemical and ecotoxicological parameters, the importance of which has recently been recognised by EU regulatory dispositions and OSPAR indications. Acute and sub-chronic endpoints were both considered on a whole effluent toxicity basis by means of Vibrio fischeri and Crassostrea gigas, respectively. The two months monitoring survey evidenced that the UF-MBR was more efficient than the AS-SBR in providing high-quality discharges under both chemical and ecotoxicological viewpoints.