The cultivation of microalgae using urban wastewater as a nutrient substrate represents a promising bio-refinery concept that can serve multiple purposes; indeed, it allows for the generation of biomass, which can be used for various applications while meanwhile removing nutrients from wastewater. In this study, the potential of urban wastewater collected at two different time periods in a farmhouse as a nutrient substrate for microalgal growth was assessed. Wastewater samples were treated on a laboratory scale, inoculating reactors with two common species, Chlorella vulgaris (CV) and Scenedesmus quadricauda (SQ), and with an autochthonous strain of Klebsormidium sp. K39 (Kleb), directly isolated from effluents of the same system. The main aim of the study was to compare the microalgae’s performances in terms of wastewater re-mediation and biomass productivity. In the first case study, which involved an effluent with a lower pollutant level, microalgal cultivation showed removal efficiencies in the range of 57–63% for total nitrogen, 65–92% for total phosphorous, 94–95% for COD, and 100% for E. coli. In the second case study, involving an effluent with a higher pollutant level, the remediation performances of the three microalgae strains ranged from 93 to 96% for total nitrogen, from 62 to 74% for total phosphorous, from 96 to 97% for COD, and 100% for E. coli. At the end of the experimental trials, treated waters showed values of pollutants suitable for irrigation use, in accordance with environmental and national legislation, which established specific thresholds for irrigation purposes.