Fusarium wilt, caused by Fusarium oxysporum, is a devastating soilborne fungal disease. Compared to traditional chemical pesticides, biological control has been attracting increasing attention recently, owing to its eco-friendly features. This study investigated the potential use of mixed culture fermentation of Rhizopus nigricans and Trichoderma pseudokoningii (MCF) to control Fusarium oxysporum f.sp.cucumerinum (FOC). At first, the tablet confrontation experiment using bio-control fungi and F. oxysporum showed that the suppression rate of R. nigricans and T. pseudokoningii were more than 60% higher than that of individual fermentations. Furthermore, the inhibitory effect of MCF on the pathogen was higher than that of single fermentation broth, both in vitro and in greenhouse experiment. At the molecular level, MCF had better control on the number of pathogens in the plant tissue and soil, as seen by the real-time PCR assay. In the pot experiment, increased activities of the defense enzymes involved in PAL, SOD, and POD indicated that mixed broth treatment could increase the plant-induced resistance. In the field experiment, after two-year treatment by the mixed broth, the relative control effect on cucumber Fusarium wilt was 76.5%. Through this study, we demonstrated that co-fermentation of R. nigricans and T. pseudokoningii has synergistic effect on the control of F. oxysporum, which may promote more reasonable and effective utilization of biocontrol agents in future. •We used mixed culture fermentation (MCF) for biological control of Fusarium wilt.•MCF of Rhizopus nigricans and Trichoderma pseudokoningii was used.•The relative control effect on cucumber Fusarium wilt was 76.5%.•Co-fermentation of R. nigricans and T. pseudokoningii showed a synergistic effect.