Five treatments were applied: control (CK), reductive soil disinfestation (RSD) using alfalfa as organic material (RSD0), liming before RSD0 (RSD1), adding potassium sulfate before RSD0 (RSD2), and adding lime and potassium sulfate before RSD0 (RSD3). Then, RSD-treated soils were inoculated with Fusarium oxysporum or not inoculated, and control soils were not inoculated. Cucumber was then cultivated for one season in all treatments. Soil pH increased after RSD treatments and then decreased after cucumber harvesting. Soil SO4 2− content decreased after RSD treatments but then returned to its initial level during cultivation. The abundance of F. oxysporum significantly decreased after all RSD treatments, but it increased in the liming and high SO4 2− content RSD treatments (RSD1–3) over the course of the growing season. The cucumber plants were infected by the pathogen in the CK and F. oxysporum-inoculated RSD treatments with high SO4 2− content but were not infected in the RSD treatment without SO4 2− addition. The RSD treatment without liming or SO4 2− addition had the lowest relative abundance of potential pathogens (i.e., Fusarium and Penicillium) and the highest relative abundance of potential antagonists (i.e., Chaetomium). In conclusion, soil microbial biomass increased, and microbial community composition changed to become more suppressive to pathogens after the RSD treatment. The high SO4 2− content influenced the microbial composition and weakened the ability of the soil treated with RSD to suppress disease.