Humans can be potentially exposed to Enterocytozoon bieneusi and Encephalitozoon spp. from topsoils when playing or spending time in recreational areas. 227 topsoil samples were collected across Alcalá de Henares (Spain) in July 2017: 155 urban, 60 industrial and 12 from a public garden. Simultaneous detection was performed using a SYBR Green real-time PCR following appropriate extraction of DNA with Fast-Prep for Soil^®. Organic matter content (OM), pH, electric conductivity (EC) and soil texture (percentages of sand, clay and silt) were also determined. E. bieneusi was detected in 9 (5 urban, 4 industrial) meanwhile Encephalitozoon spp. in 22 topsoil samples (16 urban, 6 industrial; ten E. intestinalis, nine E. intestinalis/E. hellem and three E. cuniculi). The presence E. bieneusi was associated within urban soils that presented lower EC for (0.50 vs 0.71; p-value=0.011), as this factor may provide a richer environment for the survival of the spores. The presence of microsporidian spores where higher in those topsoils with higher OM, for E. bieneusi (6.96 vs. 4.98; p-value=0.034) and E. intestinalis/E. hellem (5.53 vs. 3.12; p-value=0.0007). E. intestinalis was significantly higher in topsoils with lower content of sand (14.5 vs 21.74; p-value=0.00003) and clay (11 vs. 19.02; p-value=0.020), but higher content of silt (78.5 vs 64.95; p-value=0.023), which might be attributed to the differences in the capacity of topsoils in retain moisture in function of their texture. A better understanding of these potential associations is critical to select appropriate decontamination techniques and strategies to prevent and minimise human exposure.