Municipal wastewater treatment plants play a vital role in reducing the microbial load of sewage before the end-products are discharged to surface waters (final effluent) or local environments (biosolids). This study was to investigate the presence of human-virulent microsporidian spores (Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon hellem) and enterococci during treatment processes at four Irish municipal secondary wastewater treatment plants (plants A-D). Microsporidian abundance was significantly related to seasonal increase in water temperature. Plant A had the least efficient removal of E. intestinalis spores (32%) in wastewater, with almost 100% removal at other plants both in April and July. Some negative removal efficiencies were obtained for E. bieneusi (at plants C and D, -100%) and for E. hellem (at plants A and D, -90% and -50%). In addition, a positive correlation was found between the levels of enterococci and E. bieneusi in July (r (s) = 0.72, P < 0.05). In terms of the dewatered biosolids, a median concentration as high as 32,000 spores/Kg of E. hellem was observed at plant D in July. Plant C sewage sludge contained the lowest microsporidian loadings (E. bieneusi; 450 spores/L and 1,000 spores/L in April and July, respectively). This study highlights the seasonal variation in concentrations of microsporidian spores in the incoming sewage. Spores in final effluents and dewatered biosolids can be the source of human-virulent microsporidian contamination to the local environment. This emphasizes a considerably high public health risk when sewage-derived biosolids are spread during summer months. This study also suggested enterococci as a potential indicator of the presence of microsporidian spores in wastewater, especially for E. bieneusi.