The invention discloses a method for determining an antibacterial mechanism of a zeolite molecular sieve antibacterial agent by high performance liquid chromatography. The zeolite molecular sieve antibacterial agent is taken as a research object, escherichia coli and staphylococcus aureus are taken as indicator bacteria, and content changes of staphylococcus aureus mycoprotein and content of beta-lactamase in escherichia coli after action of the antibacterial agent are determined by means of high performance liquid chromatography to realize expounding of the antibacterial mechanism of the zeolite molecular sieve antibacterial agent. The method is simple in operation, high in sensitivity, low in sample consumption and quick in detection. Along with increase of the concentration of the antibacterial agent, HPLC (high performance liquid chromatography) peak area is reduced, and protein content is decreased, so that the bacterial structure is changed and even completely damaged, bacteria fail to grow normally since cell metabolism and growth are affected, and bacterium inhibition and killing are realized.