One of the main cucumber (Cucumis sativus) diseases in China is Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. cucumerinum (FOC). The aims of this study were to evaluate the suppressive capacity of different spent mushroom substrate (SMS) amendments against Fusarium wilt and to analyse their effects on soil microbiological properties. A greenhouse experiment was carried out with four treatments: (1) CK: unamended control, (2) FV: application of spent Flammulina velutipes substrate, (3) LE: application of spent Lentinus edodes substrate, and (3) PO: application of spent Pleurotus ostreatus substrate. Cucumber disease incidence, the abundance of FOC, Bacillus, Pseudomonas and Lysobacter (using real-time PCR), and the microbial community (using Biolog EcoPlate and phospholipid fatty acid (PLFA) analyses) in rhizosphere were studied 15, 30, 45 and 60 days after transplantation. At the end of the 60-day pot experiment, the FV, LE and PO treatments significantly (p < 0.05) reduced disease incidence by 53.3%, 25.7% and 37.9%, respectively, compared to the CK treatment. All three SMS amendments significantly (p < 0.05) increased the growth of cucumber seedlings and suppressed the FOC population compared to the control. The three SMS amendments, in particular spent F. velutipes substrate, increased the abundance of beneficial microbes (Bacillus spp., Pseudomonas spp. and Lysobacter spp.). Biolog and PLFA data showed that average well colour development (AWCD), total PLFAs, bacterial, and Gram (+) and Gram (−) bacterial PLFAs were increased in the FV, LE and PO treatments. Overall, the results of this study suggest that the application of three SMS amendments, especially FV, could effectively suppress cucumber Fusarium wilt and reducing the population of FOC.