This study aimed to investigate the inhibitory effect of berberine hydrochloride (BH) on Candida albicans (C.albicans) ATCC10231 biofilm formation. This paper found a positive correlation between the concentration of BH and its inhibitory effect on the cellular activity of early biofilms because we found that 128 and 32 μg/mL BH significantly inhibited biofilm formation (P < 0.05). BH significantly inhibited the cellular activity in early biofilms, destroyed the microscopic morphology of C.albicans and reduced the thickness of the biofilm. Both 128 and 32 μg/mL concentration solutions of BH significantly inhibited biofilm formation (P < 0.05). We found that the inhibitory effect of BH solution was positively correlated with its concentration and 128 μg/mL BH was better than 4 μg/mL fluconazole. Additionally, the results of RT-PCR indicated that 128 and 32 μg/mL BH inhibited the expression of EFG1, HWP1, ECE1, and ALS1 (P < 0.05). The efficacy of BH in inhibiting the formation of C.albicans biofilm by killing the cells in the biofilm and destroying its structure; and the mechanism may be to down-regulate the expression of EFG1, HWP1, ECE1, and ALS1 in hyphae formation, thereby, retarding the morphological transformation of C. albicans.