This study investigated the effects of co-application of spent mushroom substrate (SMS) – based bio-organic fertilizer and compound fertilizer on passion fruit, soil properties, and rhizosphere microorganisms. The results demonstrated that the co-application significantly increased the total nutrients (17.73-81.21%) and available nutrients (25.09-102.80%) in the soil compared to the control group. The soil pH decreased by 0.23-0.71, and the soil organic matter content increased by 1.29-32.77%. Regarding microbial diversity, the application of SMS - based bio-organic fertilizer enhanced the abundance of beneficial microorganisms, reduced the disease incidence, and enhanced the crop’s resistance to heavy metals. Regarding yield, the co-application treatment group achieved the highest passion fruit yield of 1.27 kg/plant, which was significantly higher than the control treatment (0.42 kg/plant). Fruit quality indicators, including soluble solids, acidity, and sugar-acid ratio, were also significantly improved. Based on the integrated results of soil properties, microorganism diversity, and yield performance, it is recommended to apply 2 kg/plant of SMS - based bio-organic fertilizer as a base and 8 g/plant of compound fertilizer as top dressing in passion fruit cultivation to enhance soil quality and optimize both yield and quality.