Postharvest soft rot of kiwifruit has resulted in substantial market losses, yet there were few antagonistic yeasts reported to control the disease. This study screened 1113 yeast strains for potential antagonistic yeast to control soft rot of kiwifruit caused by Botryosphaeria dothidea and Diaporthe actinidiae, and strain 37 was selected to evaluate the control efficacy and mechanisms, which was identified as Meyerozyma guilliermondii via molecular biological identification. Our results showed that M. guilliermondii 37 effectively reduced pathogen spore germination rate to 28.52% and decay incidence of inoculated kiwifruit to 42.11% maximumly, whereas cell-free supernatant lacked antifungal activity, implying that M. guilliermondii 37 didn't produce direct antifungal compounds against the two pathogens. In addition, M. guilliermondii 37 adhered tenaciously to the pathogens' mycelium and colonized rapidly in kiwifruit flesh. Moreover, yeast strain 37 induced kiwifruit resistance by elevating the defense-related enzyme activity, increasing the antioxidant substances content, and suppressing the cell wall-degrading enzyme activity. Gene expression was consistent with the corresponding enzyme activity. Further postharvest yeast immersion treatment significantly reduced natural decay to 35.69% while maintaining soft-ripe quality. These results indicated that M. guilliermondii 37 might serve as a biocontrol agent against postharvest soft rot in kiwifruit.