In order to explore the inhibitory effects of Prunus mume, Crataegus pinnatifida and Coptis chinensis on Vibrio parahaemolyticus and its biofilm in vitro, the agar diffusion method was applied. These three Chinese herbal medicines had different inhibitory effects on V. parahaemolyticus. The inhibition zone of C. pinnatifida to V. parahaemolyticus was (15.25 ± 0.53) mm, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of C. pinnatifida on V. parahaemolyticus were both 31.25 mg/mL; the inhibition zone of C. chinensis to V. parahaemolyticus was (18.08 ± 0.10) mm, and the MIC and MBC of C. chinensis on V. parahaemolyticus were both 15.63 mg/mL; the inhibition zone of P. mume to V. parahaemolyticus was (28.99 ± 0.47) mm, and the MIC and MBC of P. mume on V. parahaemolyticus were both 7.81 mg/mL. The effects of three traditional Chinese medicines on the biofilm formation of V. parahaemolyticus were tested by MTT colorimetric method using methylthiazolyl tetrazolium (MTT). P. mume, C. pinnatifida and C. chinensis have significant inhibitory effects on V. parahaemolyticus biofilm and their MIC are 7.81 mg/mL, 3.125 mg/mL, and 62.5 mg/mL, respectively (P < 0.01). The experimental results are expected to provide certain references for the development of new fishery drugs.