Prototheca are unicellular, non-pigmented, yeast-like microalgae widely distributed in natural and anthropogenic environments. While typically saprophytic, these organisms are the only plant species known to have emerged as opportunistic pathogens for vertebrate animals including humans. The algae have been recognised as ubiquitous inhabitants of domestic and municipal sewage, considered as point sources of Prototheca contamination in natural aquatic systems. These observations, however, have been based on data from a very limited number of historical studies. The purpose of this work was to readdress the question of wastewater as a habitat for Prototheca, using a new and refined experimental methodology, and in light of the recently updated taxonomy and nomenclature of the Prototheca genus. Over a three-month period, 96 samples were collected from three municipal wastewater treatment plants, at four different stages of the treatment process. The algae were recovered from 74 samples, which translates into an overall prevalence of 78.1%. The highest Prototheca population was found in raw sewage, and decreased progressively with each purification stage. The Prototheca cell count in the effluent was 100-fold lower than in the untreated sewage, resulting in an algal removal efficiency of up to 99.9%. The occurrence of Prototheca in treated wastewater suggests that wastewater treatment plants may act as a pathway for the release of these algae into receiving natural environments. In both influent and effluent, species distribution was dominated by Prototheca species previously reported as pathogenic, with P. wickerhamii detected most frequently. The study concludes by advocating for regular monitoring of pre- and post-treatment wastewater for Prototheca species and emphasising the need for guideline limits on their acceptable levels at both treatment stages.