Reductive soil disinfestation (RSD) is an eco-friendly and effective approach to control a wide range of soil-borne pathogens. As the application of organic matter is crucial for the success of RSD, the efficacy of reductive soil disinfestation incorporated with silkworm excrement (SE) for Fusarium oxysporum suppression was evaluated. Quantitative and cultivable experiments showed that F. oxysporum declined markedly in all anaerobic treatments regardless of SE additive amount (0, 0.5%, and 1%). However, pot experiment revealed that F. oxysporum significantly decreased only in the RSD-treated soil after pepper planting, especially in the soil amended with 1% SE. Then, the dissimilarities among soil microbial communities after RSD treatment and pepper planting were investigated by MiSeq sequencing. PCoA analysis showed that RSD treatment significantly altered bacterial and fungal community structure compared with that of the flooding and control soils. The bacterial taxa UC-Rhodothermaceae, UC-BSV40, Clostridium_sensu_stricto_8, UC-Coriobacteriaceae, Microvirga, and Oxobacter, and the fungal taxa, UC-Myriangiales, Sordaria, Trichoderma, and Cutaneotrichosporon, most of which were dominant microorganisms in the RSD soil, showed negative relationships with F. oxysporum. After pepper planting, several profitable strains, such as Mortierella, Apodospora and Chrysosporium, also markedly increased in the RSD soil. Taken together, this work provides a preliminary insight into the effect of RSD incorporated with SE on pathogen suppression during the anaerobic process and the planting stage.