Abstract: Mycobacterium smegmatis, a classical model organism in tuberculosis research, exhibits characteristics of intrinsic drug resistance and clinical pathogenicity, capable of causing skin and soft tissue infections. To overcome its intrinsic resistance barrier, this study employed combined treatments of M. smegmatis with varying concentrations of n-butanol and kanamycin at different treatment durations. Bacterial viability was quantified, and propidium iodide（PI）staining was utilized to assess cell membrane integrity. Results demonstrated that the combination of 0.5 mol·L⁻¹ n-butanol with kanamycin achieved optimal synergistic bactericidal efficacy. This synergistic effect was significantly enhanced within 10 minutes of combined treatment and exhibited both concentration-independent and rapidly responsive characteristics. Furthermore, n-butanol exerted a broad-spectrum synergistic effect with both kanamycin and streptomycin. PI staining revealed significantly higher fluorescence intensity in the combination treatment group compared to single-agent treatments, indicating substantially increased cell membrane permeability. These findings provide a theoretical foundation for developing novel adjuvant therapies for anti-tuberculosis drugs.