Biodiesel Co-Product (BCP) amendment has been shown to decrease both nitrate leaching and nitrous oxide (N2O) emissions in acidic soil; however, the effects of BCP on the soil microbiome have not been investigated thoroughly. In this study, we investigated the response of prokaryotic and fungal communities in aspects of structure, diversity, and co-occurrence network to the BCP amendment following complete mixing application (0–18-cm depth) of 1.5 mg BCP-C g
1 and surface application (0–6-cm depth) of 4.5 mg BCP-C g
1 via high-throughput 16S rRNA and internal transcribed spacer (ITS) amplicon sequencing. The amendment altered microbial communities significantly by increasing the relative abundances of Proteobacteria (Burkholderia) and Ascomycota (Trichoderma) in prokaryotic and fungal communities, respectively. Only a higher rate application (4.5 mg BCP-C g
1) decreased prokaryotic alpha diversity, whereas all rates of amendment decreased fungal diversity. The co-occurrence network of prokaryotes had more nodes and links and a higher average degree and clustering coefficient than the fungal network with BCP addition. The majority of keystone species in prokaryotic and fungal networks were from Proteobacteria and Ascomycota taxa. Of note, the BCP amendment significantly increased the OTU numbers of potential biocontrol agents, including Trichoderma (T.) spirale, T. koningiopsis, and T. virens, etc., while decreased OTU numbers related to plant pathogens species, particularly in the complete mixing application. Our work highlights the potential for BCP amendments to promote the assembly of a healthy soil microbiome by enhancing the abundance of