Non-toxigenic strains of Aspergillus flavus (Link) can be used as biological control agents to reduce the contamination of grains such as maize with carcinogenic and immunotoxic aflatoxins. Based on its sclerotial morphology, A. flavus is commonly divided into two types: L strains and S strains. In this study, we applied the non-toxigenic A. flavus L strain BN030D to the soil of five maize fields in Djidja, central Benin, to investigate its effects on the composition of the prevailing fungal communities, on the ratio of A. flavus L strains and S strains, and on the vegetative compatibility of L strains with BN030D. Soil samples were taken before the strain application, and soil and cob samples were taken after BN030D treatment, i.e., at maize harvest. BN030D did not affect the composition of A. flavus, Fusarium spp., Penicillium spp. and A. niger in the soil. There was no significant difference between the number of A. flavus L and S strains in the control and (subsequent) treatment plots before treatment, but significantly more L than S strains in soil and maize after BN030D treatment. About 1% of all L strains isolated from the untreated soil samples were vegetatively compatible to BN030D, confirming the natural presence of the non-toxigenic A. flavus L strain in the study area. Additionally, 59% (soil) and 26% (maize) of all strains isolated from the treated samples belonged to the BN030D vegetative compatibility group. BN030D spread over time and space from the release area into the non-treated buffer area. The implications of these findings for strategies to reduce aflatoxin contamination of maize and the potentials of such a competitive replacement approach for biological control of A. flavus in West Africa are discussed.