BACKGROUND AND AIM: Topsoils can be a source of emerging opportunistic and zoonotic parasites. Children and immunodeficient individuals can be accidentally exposed to microsporidia spores when spending time in urban parks and recreational areas. The aim of this study was to determine the presence and distribution of human-related microsporidian species in urban/industrial areas in Alcalá de Henares (Madrid Region, Spain). METHODS: 227 topsoil samples were collected July 2017 across different urban (n=155) and industrial (n=60) areas in Alcalá, and in a public garden in the city centre (n=12). DNA was extracted from concentrated pellet by disrupting the spores using Fast-Prep for Soil, followed by SYBR Green real-time PCR for simultaneous detection of {Enterocytozoon bieneusi} and {Encephalitozoon} spp. ({E. intestinalis}, {E. hellem} and {E. cuniculi}). RESULTS:Real-time amplification with MsRTf1/MsRTr1 primer set and differential melting curve analysis confirmed presence of {E. bieneusi} (n=9; 5 urban, 4 industrial), and {Encephalitozoon} spp. in 22 of the soil samples: three {E. cuniculi} (2 urban, 1 industrial); ten {E. intestinalis} (8 urban, 2 industrial); nine {E. intestinalis}/{E. hellem} (6 urban, 3 industrial). Microsporidia spores were not found in any of the samples monitored in public gardens. Urban animals have been suggested to play a role in the distribution of these emerging parasites through their droppings. Thus, similar studies carried out in Madrid city have detected microsporidia in animal faeces collected from public parks but not in their soils, which highlights the significance of our study. CONCLUSIONS:Our results indicate a moderate prevalence and distribution of human-related microsporidia in soils in Alcalá, which could represent a public health concern. Interventions to protect the public might be necessary as the presence/distribution of these biothreats could be higher, as potential false-negative results due to low DNA parasite concentration and/or the presence of environmental PCR inhibitors should be considered. KEYWORDS: Microsporidia, urban topsoils, human risks, biological contamination