The effect of fungichromin (FC) on the formation of Candida albicans biofilm was assessed using 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2 H -tetrazolium-5-carboxanilide (XTT) reduction method, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Results showed that FC revealed an inhibitory effect on the formation of C. albicans biofilm in a dose-dependent manner with a minimum inhibitory concentration (MIC) of 10 µg/mL. Over 80% of biofilm formation was inhibited by FC at the concentration of 40 µg/mL when compared with the control. Similarly, real-time PCR showed that the expression of the genes such as ALS1 , ALS3 , HWP1 , EFG1 , HYR1 , CPH1 and BCR1 appeared to be remarkably affected by FC at the concentration of 20 µg/mL during the biofilm formation. In addition, FC could also induce the apoptosis of C. albicans cells in a dose-dependent manner. Therefore, FC displayed potent activity against the formation of C. albicans biofilm in vitro and played an important role in reducing the incidence of device-associated infections.