Bovine respiratory diseases are a major problem for animals and are caused by Pasteurella multocida, commonly found in animals' respiratory tracts. Pasteurella multocida is considered as a secondary pathogen, and the epidemiology of this bacteria related to the respiratory tract poorly investigated yet. By use of high antimicrobial drugs in the clinics, coupled with the fact that the biofilm formed by Pasteurella multocida is conducive to increase its resistance to unfavourable environment, the resistance of Pasteurella multocida in the bovine respiratory tract is becoming increasingly serious, which poses a certain challenge to the treatment of bovine respiratory diseases. In this study, the Kirby Bauer (K.B) disk diffusion method was used to conduct in vitro drug susceptibility testing of 20 antimicrobial drugs on 15 isolates of Pasteurella multocida; PCR detected the carriage of relevant resistance genes of the isolates; the crystal violet staining method was used to determine the ability of the isolates to form biofilm, and the effects of thymol and berberine on the adherence value (B-value) of the biofilm of the isolates and their growth curves; laser confocal microscopy was used to observe the effects of thymol and berberine on the formation of biofilm; the effects of thymol and berberine on the expression of genes luxS, fliA, and motA related to biofilm formation were detected by qRT-PCR. The results showed that all 15 isolates showed 100% sensitivity to tilmicosin, ceftriaxone, ampicillin/sulbactam, and florfenicol, and 53.3% resistance to erythromycin; the resistance rates to lincomycin, clindamycin, and streptomycin were 93.3, 46.7, and 40%, respectively. The aminoglycoside resistance genes strA and strB had the highest detection rates of 93.3 and 80%, respectively, followed by the macrolide resistance gene mefE with a detection rate of 66.7% and the β-lactam resistance gene blaROB-1 with a detection rate of 60%. There were 13 biofilm-positive isolates, accounting for 86.7% of the total. The Thymol MIC group, berberine MIC group, and 1/2 MIC group could inhibit the formation and growth of the strain's biofilm in a concentration-dependent manner. Thymol MIC, berberine MIC, and 1/2 MIC groups all significantly down-regulated the expression of luxS, fliA, and motA, genes related to biofilm formation. The results of this study provide medication references and theoretical guidance for the prevention and treatment of bovine respiratory diseases caused by biofilm-positive Pasteurella multocida infections in clinics and reduce the resulting economic losses.