Antibiotics are widely used as the most effective treatment for bacterial infections. However, the extensive use of antibiotics in clinical practice has led to elevated levels of bacterial antibiotic resistance in common clinical pathogens. One potential solution to this problem is the use of traditional Chinese herbal medicines to supplement antibiotic effectiveness. In this study, we utilized the matrine derived from the leguminous shrub Sophora flavescens and examined synergism with 17 clinically relevant antibiotics against Staphylococcus aureus, β-hemolytic Streptococcus, Pasteurella multocida, Escherichia coli and Salmonella paratyphi B using in vitro tests. Matrine exhibited significant antibacterial activity against these bacteria with minimum inhibitory concentrations (MIC) ranging from 4.69 to 9.38 mg/mL. Matrine combinations also yielded fractional inhibitory index values between 0.14 and 1 indicating additive or synergistic effects without antagonism. Furthermore, the mutation prevention concentration (MPC) analysis revealed that matrine could mitigate the impact of ceftiofur, doxycycline, gentamicin and tilmicosin for all 5 bacterial strains. The reduced MPC and MPC/MIC values of these antibiotics demonstrated a narrower selection window for drug-resistance mutations thereby retarding the development of drug resistance. The combination of matrine and antibiotics thereby enhanced the antibacterial activity of all test antibiotics while reducing the antibacterial impact of ceftiofur, doxycycline, gentamicin and tilmicosin on S. aureus and β-hemolytic Streptococcus. Additionally, matrine combinations with the latter drugs could lower the anti-mutation concentration thereby reducing the emergence of drug resistant strains.