Fusarium wilt is one of the main diseases of cucumber, and bio-organic fertilizer has been used to control Fusarium wilt. In this study, a pot experiment was conducted to evaluate the effects of bio-organic fertilizer applied at four levels on the suppression of Fusarium wilt disease in cucumber, the soil physico-chemical properties and the microbial communities. In comparison with the control (CK), low concentrations of bio-organic fertilizer (BIO2.5 and BIO5) did not effectively reduce the disease incidence and had little effect on soil microorganisms. High concentrations of bio-organic fertilizer (BIO10 and BIO20) significantly reduced the disease incidence by 33.3%-66.7% and the production was significantly improved by 83.8%-100.3%. The soil population of F. oxysporum f. sp. cucumerinum was significantly lower in bio-organic fertilizer treatments, especially in BIO10 and BIO20. The microorganism activity increased with the bio-organic fertilizer concentration. High-throughput sequencing demonstrated that, at the order level, Sphingomonadales, Bacillales, Solibacterales and Xylariales were significantly abundant in BIO10 and BIO20 soils. At the genus level, the abundance and composition of bacterial and fungal communities in BIO10 and BIO20 were similar, illustrating that high concentrations of bio-organic fertilizer activated diverse groups of microorganisms. Redundancy analysis (RDA) showed that Xanthomonadales, Sphingomonadales, Bacillales, Orbiliales, Sordariales, and Mucorales occurred predominantly in the BIO10 and BIO20. These microorganisms were related to the organic matter, available potassium and available phosphorus contents. In conclusion, a high concentration of bio-organic fertilizer application suppressed the Fusarium wilt disease and increased cucumber production after continuous cropping might through improving soil chemical condition and manipulating the composition of soil microbial community.