Objective This study aimed to investigate the inhibitory effect of berberine hydrochloride (BH) onCandida albicans(C.albicans) ATCC10231 biofilm formation. Results 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 mu g/mL BH significantly inhibited biofilm formation (P < 0.05). BH significantly inhibited the cellular activity in early biofilms, destroyed the microscopic morphology ofC.albicansand reduced the thickness of the biofilm. Both 128 and 32 mu g/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 mu g/mL BH was better than 4 mu g/mL fluconazole. Additionally, the results of RT-PCR indicated that 128 and 32 mu g/mL BH inhibited the expression ofEFG1,HWP1,ECE1, andALS1(P < 0.05). Conclusion The efficacy of BH in inhibiting the formation ofC.albicansbiofilm by killing the cells in the biofilm and destroying its structure; and the mechanism may be to down-regulate the expression ofEFG1,HWP1,ECE1, andALS1in hyphae formation, thereby, retarding the morphological transformation ofC. albicans.