Macrophomina phaseolina (Tassi) Goid. is a soilborne fungus that causes charcoal rot in numerous crop plants, reducing yield and profits. This study assessed the efficacy of pre-season mustard cover crops (Brassica juncea), a chemical fungicide seed treatment, a combination of both, and a non-treated control in a set of varieties that ranged in maturity. In 2016 and 2017, 'AG4135 RR2Y/SR' consistently yielded lower compared to other varieties. Early maturity varieties exhibited reduced yields relative to mid and late maturity pairs in 2016 and 2017. Root colonization was influenced by treatment, with the combined pre-season cover crop and seed treatment exhibiting the lowest colonization rates in 2015, while all treatments had significantly reduced colonization in 2017 compared to untreated controls. Additionally, differences in root colonization were observed among soybean varieties, suggesting genotype-specific responses to disease management strategies. M. phaseolina soil populations showed a marked increase from pre-cover crop to post-soybean harvest underscoring the pathogen's proliferation throughout the growing season. Correlation analyses revealed significant negative relationships between root colonization and yield for untreated controls, suggesting that higher root colonization adversely affected crop performance. These findings suggest that specific treatments may help mitigate M. phaseolina population build-up in roots and soil and indicate that the effectiveness of control measures may be influenced by pathogen variability in response to environmental conditions and management practices. These findings also contribute to our understanding of effective strategies for managing charcoal rot disease and highlights the importance of integrating chemical and biological control methods.