There is a growing consensus that plant-derived antimicrobials may be a safe and effective alternative to synthetic chemical preservatives against foodborne pathogens. This study aims to investigate the potential application of the stems and leaves of Astragalus membranaceus (AMSL) in food preservation. Antibacterial activity of AMSL was evaluated by the disk diffusion method and the minimum inhibitory concentration (MIC) assay, and then the activity stability at different conditions (temperatures, pHs and ultraviolet irradiations) was analyzed and the antibacterial mechanism was revealed. Chemical composition of the active substances was also identified by UPLC–MS/MS. The results showed that the extracts from AMSL had low activities against Geotrichum candidum and Escherichia coli, while good action on Staphylococcus aureus, Bacillus subtilis, Aspergillus flavus, and Candida albicans. In contrast, the ethyl acetate fraction (EAF) of AMSL was the most active against Bacillus subtilis with a diameter of inhibition zone of 15.75 mm and the MIC value of 12.5 mg/mL. EAF exhibited good stability against heating at 80°C for 30 min, 60 min exposure to UV light, and it was effective in pH range of 2.0–6.0. After treating by EAF, the growth of the Bacillus subtilis was inhibited with delayed logarithmic phase, and the cell wall and the cell membrane of the bacteria were damaged deduced from the increased extracellular activity of alkaline phosphatase, the leakage of nucleic acids, and the increase of extracellular conductivity. Further SEM and TEM analysis confirmed the destroying of the cell wall integrity and cell membrane permeability with visually wrinkled even empty cells caused by EAF. Subsequent analysis revealed that flavonoids were the predominant components of EAF, followed by organic acids. Our findings provided a theoretical possibility that the AMSL could be used as a natural food preservative in the food industry.