Apple ring rot, caused by Botryosphaeria dothidea, is one of the important diseases in China. This pathogen infects branches and fruit and also results in fruit decay during storage. Biocontrol agents have been proposed to reduce apple decays during storage and are considered as a promising alternative strategy to traditional chemical treatment. In this study, Meyerozyma guilliermondii Y-1, isolated from healthy grape fruit, was firstly evaluated for its biocontrol efficiency against B. dothidea in postharvest apple fruit, and the possible mechanisms were investigated. The results revealed that M. guilliermondii Y-1 treatment effectively reduced apple ring rot caused by B. dothidea in vivo. The disease incidence and lesion diameter were reduced by 32.22% and 57.51% compared with those of control fruit. Furthermore, the use of filtrate and autoclaved culture of M. guilliermondii Y-1 also showed a certain degree of control efficiency against fruit ring rot. M. guilliermondii Y-1 significantly inhibited the mycelial growth and spore generation of B. dothidea in vitro and exhibited an obvious ability to colonize in apple fruit wounds and surface at 25 °C or 4 °C. In addition, M. guilliermondii Y-1 treatment significantly enhanced the activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonialyase (PAL), and polyphenoloxidase (PPO), promoted the total phenolics content, and alleviated lipid peroxidation in apple fruit. As expected, we found that the expression of four pathogenesis-related proteins genes (MdPR1, MdPR5, MdGLU, and MdCHI) was remarkably increased by M. guilliermondii Y-1 treatment. Our data together suggest that M. guilliermondii Y-1 is a potential biocontrol agent against B. dothidea postharvest infection in apple fruit, partially through inhibiting mycelial growth and spore germination of B. dothidea, competing for space and nutrient with pathogen, and inducing resistance in apple fruit by stimulating a series of defense responses.