Zambia is one of the African countries failing to export groundnuts to restricted markets because of high aflatoxin concentrations. Currently, the volume of groundnuts being exported has significantly reduced compared to the period between 1960 and 1970. Since aflatoxin formation starts in the field, aflatoxin mitigation measures must also begin at field-level. A greenhouse experiment was conducted to determine the interactive effects of water stress and exchangeable calcium on pre-harvest aflatoxin content in kernels. Four water moisture levels combined with four levels of gypsum application were applied from flowering to full maturity. The groundnuts were harvested at full maturity and the total biomass and aflatoxin content in kernels were determined. Treatment effects were determined by Analysis of Variance and the non-parametric Kruskal Wallis test at 95% Confidence Interval. Water stress resulted in about 10 folds’ increase in aflatoxin content at the least level of water supply (p = 0.032). There were no significant differences in the distribution of mean pre-harvest total aflatoxin concentrations due to gypsum application (p = 0.274). Similarly, there were no significant differences in the distribution of mean total biomass attributed to both water stress and gypsum application. These results suggest that adequate water supply can help to minimize pre-harvest aflatoxin incidences, while the time and the rates of gypsum applied as a source of calcium may also influence the aflatoxin response to the treatment.