In a previous study on discovering new antimicrobial agents from microbial sources, nine bafilomycins were isolated from the fermentation broth of Streptomyces albolongus. Among them, bafilomycin C1 (Baf C1) showed strong antifungal activity against Candida albicans, with MIC value of 1.56 μg/mL. The aim of this study was to evaluate the action mechanism of Baf C1 against C. albicans. Quantitative PCR analysis revealed that ergosterol biosynthesis-related genes of C. albicans ACS1, HMG1, IDI1, ERG1, ERG2, ERG6, ERG7, ERG8, ERG9, ERG12, ERG13, ERG20, ERG24, ERG251, ERG252, ERG26, ERG27, and ERG28 were all down-regulated (Log2fold change < -1) after Baf C1(4 μg/mL) exposure. Moreover, the expression of MET6 gene, encoded methionine synthase, was also down-regulated (2.7-fold). It is corresponding with the quantitative PCR result, the content of ergosterol has dropped about 41% compared with the control. Transmission electron microscope examination also revealed that the Baf C1 strongly destroyed the cell membrane of C. albicans. In addition, the content of farnesol was significantly increased, about 2.1-fold compared with the control. The results indicated Baf C1 caused aberrations in sterol biosynthesis, leaded to the lack of ergosterol of the fungal membrane.