Objective To investigate whether alpha-bisabolol can effectively reverse ATP binding cassette subfamily B member 1 (ABCB1)-mediated multidrug resistance (MDR) in tumors and to explore the underlying mechanisms. Methods The network pharmacology was utilized to analyze the active components of Agrimonia Eupatoria, predict target genes, and perform GO and KEGG pathway analysis. Molecular docking and thermal stability assays were conducted to examine the interaction between alpha-bisabolol and ABCB1. The MTT assay was used to assess whether alpha-bisabolol can reverse ABCB1-mediated MDR. Mechanistic studies were performed using Western blot, immunofluorescence, and flow cytometry to explore how alpha-bisabolol reverses ABCB1-mediated MDR. Results Network pharmacology indicated that alpha-bisabolol, an active compound in Agrimonia Eupatoria, is closely related to tumorigenesis. Molecular docking and thermal stability assays showed that alpha-bisabolol forms a stable complex with ABCB1. Reversal assays demonstrated that alpha-bisabolol effectively reverses ABCB1-mediated MDR in tumor cells. Mechanistically, alpha-bisabolol did not affect the expression or localization of ABCB1 in tumor cells but inhibited its excretion function, thereby increasing the intracellular drug accumulation and effectively reversing the MDR in tumor cells. Conclusion Alpha-bisabolol effectively reverses ABCB1-mediated MDR in tumor cells, offering a potential new therapeutic strategy for tumor patients with chemotherapy resistance.