Aspergillus section Flavi is able to produce aflatoxins (AFs) in vitro down to 0.85 aw with a potential maximum occurring between 0.95 and 0.99 aw, while in the field AFs increased significantly with kernel humidity below 0.95 aw. In order to clarify this apparent discrepancy, a 3-year field trial with artificial inoculation of maize ears with Aspergillus flavus strains was organised. The co-occurrence of Aspergillus section Flavi and Gibberella fujikuroi species complex (Gfsc) was observed. The incidence of A. flavus was significantly influenced by the year and negatively related to Gfsc incidence. In 2012, when the highest temperature and the lowest rain were registered, aflatoxin B1 (AFB1) content was the highest and aw < 0.95 in kernels was measured early throughout the growing season. In 2013 and 2014, the temperature was lower and rain more abundant, and aw decreased below 0.95 only close to harvest and AFB1 contamination was limited. The possibility of describing/predicting reasonably well aw dynamic based on temperature, (degree day) was confirmed. With aw > 0.95, a positive correlation between AFB1 production rate and aw was found, but a negative correlation resulted with aw < 0.95. Other factors than aw play a role, but aw = 0.95 should be considered as an indicator of suitable conditions for rapid aflatoxin accumulation.