The global prevalence of Helicobacter pylori (H. pylori) infection has led to the practice of multiple treatments with antibiotics as the standard treatment strategy. However, antibiotic resistance has led to a decline in therapeutic effectiveness, prompting the exploration of innovative and eco-friendly antimicrobial agents. Microbial biosurfactants have gained attention because of their antibacterial activities, making them a good choice for treating various bacterial infections. Therefore, our study aimed to identify active biosurfactants, their potential targets, and the accompanying signaling pathways for the therapy of H. pylori using a network pharmacology approach. We selected twenty biosurfactants from the literature for further investigation. Our analysis identified 119 potential therapeutic targets for H. pylori infection out of the 706 putative targets of biosurfactants and the 913 disease-related targets. GO analysis revealed that these 119 targets were significantly enriched into multiple GO functional categories, such as cell proliferation, and inflammatory response regulation were prominently impacted. Further, through KEGG analysis, signaling pathways, such as the HIF-1 signaling pathway, coronavirus infection, and epithelial cell signaling in H. pylori infection. Protein-protein interaction analysis further revealed 25 core targets as potential targets in the network. Molecular docking was employed to validate the efficacy of biosurfactants against the supposed targets. Remarkably, we observed that biosurfactants like athrofactin, liposan, lichenysin, sophorolipid, and amphisin exhibited strong binding affinities for the proteins EGFR, SRC, MAPK14, JUN, CXCL8, and CASP3, which are participating in the epithelial cell signaling pathway in H. pylori infection. These biosurfactants hold potential as therapeutic targets for treating H. pylori infection. Overall, the integrated approach of network pharmacology and docking analysis has been instrumental in advancing our understanding of biosurfactants potential therapeutic role in combating H. pylori infection.