Aflatoxins, produced by several Aspergillus section Flavi species in various crops, are a significant public health risk and a barrier to trade and development. In sub-Saharan Africa, maize and groundnut are particularly vulnerable to aflatoxin contamination. Aflasafe, a registered aflatoxin biocontrol product, utilizes atoxigenic A. flavus genotypes native to Nigeria to displace aflatoxin producers and mitigate aflatoxin contamination. Aflasafe was evaluated in farmers’ fields for 3 years, under various regimens, to quantify carry-over of the biocontrol active ingredient genotypes. Nine maize fields were each treated either continuously for 3 years, the first two successive years, in year 1 and year 3, or once during the first year. For each treated field, a nearby untreated field was monitored. Aflatoxins were quantified in grain at harvest and after simulated poor storage. Biocontrol efficacy and frequencies of the active ingredient genotypes decreased in the absence of annual treatment. Maize treated consecutively for 2 or 3 years had significantly (p