Despite the significance of maize (Zea mays L.) in Tanzania, Aflatoxin contamination poses significant risks to food and nutrition security, human health, and economic losses. Aflatoxin emanates from farms and farming systems which are managed by small-scale resource poor farmers. Virtually such production conditions favor predominance of Aflatoxins in the food and feed systems. However, relatively little is known about contaminants relationship on the soil ecosystems. The current study explored correlation of soil physio-chemical characteristics and aflatoxin-producing fungal species, particularly A. flavus and A. parasiticus, in maize-growing regions of Tanzania. Soil samples were collected from seven districts of Babati and Kiteto (Manyara region), Chemba, Kondoa and Bahi (Dodoma region) and Nzega and Urambo (Tabora region) previously reported high level of contamination and analyzed for physio-chemical parameters. The macro-morphological identification method was used for fungal identification from soil samples. Results exposed sandy loam soil texture was dominant across districts, low proportions of clay particles and silt. Soil chemical properties differed significantly at (P ≤ 0.001) for pH, organic matter, Total N, S, B, and EC, implicating that soil fertility status were diverse among studied districts. The correlations between soil characteristics and fungal prevalence revealed a significant correlation between certain soil physio-chemical parameters and aflatoxin-producing fungal abundance in maize agro-ecosystem. Two species namely A. flavus (38.1%) and A. parasiticus (22.2%) were dominant genera from soil samples compared to maize samples hence source of inoculums being from soil. These findings underscore the importance of soil management practices in mitigating aflatoxin contamination in maize.