Background: Rice spikelet rot disease (RSRD) is an emerging disease that significantly reduces rice yield and quality. In this study, we evaluated the potential use of the broad-spectrum endophytic fungus Phomopsis liquidambaris B3 as a biocontrol agent against RSRD. We also compared the control effects of different treatments, including chemical fungicides and treatment with multiple strains and single strains in combination or individually, against RSRD. The objective of this study was to find an effective and environmentally friendly control strategy to reduce the occurrence of RSRD and improve the rice yield., Results: In pot experiments, the effect of B3 alone was better than that of fungicide or combined measures. The results showed that root colonization by B3 significantly reduced the incidence and disease index of RSRD by 41.0% and 53.8%, respectively. This was related to enhanced superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) activity, and to significantly upregulated expression levels of OsAOX, OsLOX, OsPAL, and OsPR10 in rice. Moreover, B3 improved the diversity of the bacterial community rather than the fungal community in the rice rhizosphere. It also led to a decrease in Fusarium proliferatum colonization and fumonisin content in the grain. Finally, root development was markedly promoted after B3 inoculation, and the yield improved by 48.60%. The result of field experiments showed that the incidence of RSRD and the fumonisin content were observably reduced in rice receiving B3, by 24.41% and 37.87%, respectively., Conclusion: The endophytic fungus Phomopsis liquidambaris B3 may become an effective tool to relieve rice spikelet rot disease. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)