In recent years, with the intensification of research on antimicrobial agents, the adverse effects of antibiotics and the escalating issue of antibiotic resistance have become increasingly prominent. Prolonged antibiotic usage can induce high levels of drug resistance in certain pathogenic microorganisms, thereby complicating clinical treatment. Against this backdrop, traditional Chinese medicine (TCM) has emerged as a promising resource for combating antibiotic-resistant bacterial infections due to its natural composition, relatively low side-effect profile, and minimal potential for inducing resistance. Consequently, research on TCM has attracted widespread attention and has become a focal point in antimicrobial studies. To evaluate the in vitro antibacterial activities of water extracts and their combined formulations from seven traditional Chinese herbs, Pomegranate peel, Scutellaria baicalensis, Fraxini cortex, Eucalyptus leaves, Artemisiae argyi, Scrophularia ningpoensis, and Paeonia lactiflora, against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Enterobacter aerogenes, and Micrococcus luteus, the HPLC method was employed to quantify the content of active antibacterial compounds in each herb. Antibacterial efficacy was assessed using the filter paper disc diffusion method to determine inhibition zone diameters, and minimum inhibitory concentrations (MICs) were measured through the two-fold serial dilution technique. Based on these results, optimal antibacterial combinations were identified. Quantitative analysis revealed that Paeoniflorin in P. lactiflora was 16.80 mg/g, Baicalin in S. baicalensis reached 90.20 mg/g, and Aesculin in F. cortex was 9.16 mg/g. Chlorogenic acid, present in both Eucalyptus leaves and A. argyi, was measured at 4.66 and 3.54 mg/g, respectively. Punicalagin in Pomegranate peel was 22.18 mg/g, whereas Picfeltarraenin IA in S. ningpoensis was 1.70 mg/g. Regarding antibacterial activity, P. lactiflora and S. ningpoensis inhibited E. coli, with inhibition zone diameters of 12.2 and 11.8 mm, respectively. Pomegranate peel, Eucalyptus leaves, S. baicalensis, and P. lactiflora suppressed E. aerogenes, producing inhibition zones of 20.8, 17.2, 10.1 and 10.0 mm, respectively. Pomegranate peel and S. baicalensis exhibited strong inhibitory effects against S. aureus, with inhibition zones of 20.8 and 14.7 mm, sharing an MIC of 250 mg/mL. Against M. luteus, Pomegranate peel and Eucalyptus leaves demonstrated pronounced activity, with inhibition zones of 25.4 and 19.2 mm, and MICs of 125 and 1000 mg/mL, respectively. Notably, the combination of Pomegranate peel and S. baicalensis displayed a synergistic effect against S. aureus, with an optimal formulation ratio of 2:1. In contrast, the Pomegranate peel–Eucalyptus leaves combination synergistically inhibited M. luteus at the same 2:1 ratio. These findings underscored the potential of TCM combinations to enhance the inhibition of antibiotic-resistant bacterial strains. This study provided a robust foundation for developing novel natural antimicrobial agents and offered fresh insights and strategies to combat antibiotic resistance, highlighting both their scientific significance and practical application potential.
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