Bio-organic fertilizers (BOFs) combine functional microbes with a suitable substrate and have been shown to effectively suppress soil-borne diseases and promote plant growth. Here, we developed a novel bio-organic fertilizer (BOF) by fermentation of a cow plus chicken manure (M) compost using Fen-liquor Daqu (FLD) as a fermentation starter and compared the compositions of bacterial and fungal communities in the rhizosphere soil of watermelon plants after treatment with different fertilizers. Further, we aimed to explore the mechanisms underlying plant-promoting and disease (Fusarium wilt)-suppressing activities of each rhizosphere microbial community. The microbial communities of soil amended with cow plus chicken manure compost (S+M), soil amended with the BOF (S+BOF), and untreated control soil (S) without plants were analyzed through sequence analysis using the Illumina MiSeq platform. The results showed that a new microbial community was formed in the manure compost after fermentation by the Daqu. Application of the BOF to the soil induced remarkable changes in the rhizosphere microbial communities, with increased bacterial diversity and decreased fungal diversity. Most importantly, S+BOF showed the lowest abundance of Fusarium. Moreover, watermelon quality was higher (P < 0.05) in the S+BOF than in the S+M treatment. Thus, application of the BOF favorably altered the composition of the rhizosphere microbial community, suppressing Fusarium wilt disease and promoting plant quality.