Microsporidian spores are emerging opportunistic disease agents that are classified as worldwide diseases. In tropical areas, diagnosis of microsporidiosis is still neglected, while it may be associated with water and causes diarrhea. The evaluation of the distribution of microsporidian spores in groundwater of sub-urban areas of the Centre region of Cameroon has been our main objective. This study was conducted from August 2018 to August 2019. Microsporidian spores were identified by Weber’s stain and were observed using the Olympus CK2 inverted microscopy with immersion oil at objective 100 ×. The abundance of microsporidian spores according to species rang from classes is given as follow; 188 ([2.5–4] × [1.8–3])1, 247 ([4.5–6] × [3–5])2, 286 ([5–6] × [4–5])3, 190 ([7–8] × [4–6])4, 88 ([8–9] × [5–7])5, 44 ([9–10] × [3–4])6, 33 ([8–12] × [5–7])7, 120 ([7–11] × [4–8])8 μm with an average density of 1 ± 1 spore/10 mL of 256 samples. The density of spores can highlight epidemiologically study about microsporidia, and of the control of drinking water. The presence of human-pathogenic microsporidia in groundwater may suggest the presence of environmental reservoirs that include animals. Densities of spores were also higher during the short rainy season showing the implication of rain in the distribution of environmental spores. Statistical analysis shows that abundance of microsporidian spores increases with organic matter, turbidity and suspended solid and decreases with the temperature and total dissolved solid (P < 0.05). These results show that microsporidia may be waterborne pathogens and that population may be exposed of health risk.