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Background: In recent years, the number of incidences of dairy cow mastitis caused by non-albicans Candida (NAC) have increased owing to the virulence factors, including cell surface hydrophobicity (CSH) and phospholipase activity, of the causative agents, namely, Candida krusei and Candida parasitica. Temperature and pH changes in the cow's udder after fungal infection and unreasonable medication can affect the antifungal susceptibility of Candida spp. and their expression of virulence factors. Methods: In this study, the effects of different temperatures and pH on the virulence of NAC strains were tested, and the in vitro susceptibility of the fungal strains to Cu2+ and antibacterial agents were examined. Besides, the changes in the virulence factors of Candida spp., including biphasicity, hemolytic activity, CSH, and phospholipase activity under these test conditions were investigated, and the internal relationship between these factors was analyzed. Results: The results showed that the virulence factors and antifungal susceptibility of Candida spp. could be altered through changes in various physiological conditions. Both temperature and pH were noted to be important factors affecting Candida growth, antifungal susceptibility, and expression of virulence factors. Cu2+ inhibited the growth and virulence factors expression of Candida spp., whereas antibacterial agents directly promoted the growth of Candida spp., making them resistant, which is one of the reasons for breast inflammation symptoms in cows. Conclusions: These results on virulence factors, antifungal susceptibility, and physiological characteristics of NAC provide a theoretical basis for understanding and treating dairy cow mastitis caused by NAC.