Seeking new yeast strains having the ability to protect apple fruits against blue mould for a long time under different storage conditions was the main goal of this work. Based on the in vitro test, yeast strains KKUY0017 and KKUY0051 were selected as the most effective antagonists against Penicillium expansum. Sequencing of 26S rDNA of both yeasts confirmed that the identity of KKUY0017 and KKUY0051 was Cryptococcus albidus and Wickerhamomyces anomalus, respectively. The two strains protected the apple fruits from the blue mould disease under a wide range of temperature (5–30°C); however, W. anomalus KKUY0051 was more effective. At 25°C, W. anomalus KKUY0051 involved in the reduction of disease severity and disease incidence of blue mould by 56.49% and 57.78%, respectively. When either of the two yeasts was applied in concentration of 108 or 109 cells/mL, the maximum reduction in disease severity and disease incidence was achieved. Under cold storage (5°C), both yeast strains succeeded to protect the apple fruits free from the infection up to 24 days. Electron micrograph showed a fit attachment between the cells of C. albidus KKUY0017 and the fungal hyphae leading to the degrading of the hyphae; however, W. anomalus killed the fungal hyphae without direct attachment to them. Gas chromatography–mass spectrometry analysis of the cell-free extract of W. anomalus KKUY0051 revealed the presence of toxic compounds such as the nitrophenol derivatives. The results support the assumption that the main mode of action of this yeast is by killer toxins. We conclude that application of these yeasts under cold storage condition could keep the apple fruits free from blue mould infection for a long time.