Propionic acid (PPA) is a weak acid that has been used in food products as a preservative because of its inhibitory effect on microorganisms. In the present study, we investigated the PPA fungal killing mechanism, which showed apoptotic features. First, reactive oxygen species (ROS) accumulation and metacaspase activation were detected by 2′,7′-dichlorodihydrofluorescein diacetate and CaspACE FITC-VAD-FMK staining, respectively. Increased fluorescence intensities were observed following exposure to PPA, indicating that PPA produced an oxidative environment through the generation of ROS and activation of metacaspase, which can promote apoptosis signaling. We also examined phosphatidylserine externalization (an early apoptosis marker) and DNA and nuclear fragmentation (late apoptosis markers) after exposure to PPA. Based on the results, we determined that PPA exerts its antifungal effect by inducing apoptotic cell death. Moreover, three additional mitochondrial experiments showed mitochondrial membrane depolarization, calcium accumulation and cytochrome c release after cells were exposed to PPA, indicating that the PPA-induced apoptosis pathway is mediated by mitochondria. In conclusion, PPA induces fungal cell death through mitochondria-mediated apoptosis. Results of this study contribute to a deeper understanding of the preservative effects of PPA.