Abstract: Objective: To study the antibacterial mechanism of Goji berry by network pharmacological analysis method, and to verify the antibacterial experiment in vitro. Methods: The active components and targets of Goji berries were screened out by TCMSP, an online analysis database of traditional Chinese medicine system, and the relevant targets were searched by GeneCards and DisGeNET databases with "bacteriostatic" as the keyword. The intersection target of Goji berry and bacteriostatic was obtained through the Venny platform. The STRING database was used to construct PPI network diagrams and screen key targets in combination with Cytoscape software. GO function enrichment and KEGG pathway enrichment analysis were performed through the Metascape database. Goji berries were extracted from 80% ethanol and concentrated to extract, and the extracts of Goji berries were extracted by petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain different polar parts, and the bacteriostatic effect of different polar parts of Goji berries was determined by filter paper sheet method. Results: A total of 26 active ingredients, 368 potential drug targets, 2212 bacteriostatic targets, and 73 intersection targets of drug and bacteriostatic effect were screened. Protein interaction network analysis showed that 69 proteins, including TNF, AKT1, EGFR, HSP90AA1, PTGS2, and PPARG, might be the key targets of the bacteriostatic effect of Goji berry. GO enrichment analysis indicated that Goji berry bacteriostatic were obtained, mainly by affecting response to xenobiotic stimulus, positive regulation of gene expression, and positive regulation of nitric oxide biosynthesis process, etc. KEGG enrichment analysis showed that the antibacterial mechanism of Goji berry might be related to the pathway in cancer, C-type lectin receptor signaling pathway, lipid and atherosclerosis, etc. The results of antibacterial experiment in vitro showed that the different polar part of Goji berry extract had inhibitory effects on Staphylococcus aureus and Escherichia coli, and ethyl acetate site had the strongest inhibitory effect on Escherichia coli, and n-butanol site had the strongest inhibitory effect on Staphylococcus aureus. The minimum inhibitory concentration of ethyl acetate against Escherichia coli was 0.125 mg/mL, and the minimum inhibitory concentration of n-butanol against Staphylococcus aureus was 0.25 mg/mL. Conclusion: The extract of Goji berry could inhibit Staphylococcus aureus and Escherichia coli. Goji berry would play a role in antibacterial effect by multi-component, multi-target and multi-pathway mechanism of action.