Objective To investigate the effect and mechanism of quercetin on Epstein Barr virus (EBV)-latent membrane protein 1 (LMP1) H101R-mutated nasopharyngeal carcinoma (NPC). Methods Network pharmacology was employed to identify potential targets of quercetin in EBV-LMP1-H101R-mutated NPC. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted to explore the enriched pathways of potential targets. Molecular docking was performed to evaluate the binding affinity between quercetin and potential targets. A mutant cell model was established via lentiviral transduction, and quantitative polymerase chain reaction (qPCR) was used to detect transfection efficiency. The transfected cells were treated with 0, 20, 40, 60, 80, and 100 μmol/L quercetin for 24 h and 48 h, followed by cell counting kit 8 (CCK-8) assay to assess cell proliferation. After treatment with 0, 25, 50, and 100 μmol/L quercetin for 48 h, Transwell assay and flow cytometry were performed to evaluate cell migration and apoptosis, respectively. Western blotting was used to detect the expression of microtubule-associated protein light chain 3 (LC3) and p62 proteins after treatment with 0 and 100 μmol/L quercetin for 48 h. Furthermore, cells were treated with 0, 25, 50, and 100 μmol/L quercetin, with or without the autophagy inhibitor chloroquine (2 μmol/L) for 48 h, and cell proliferation was again measured using the CCK-8 assay. Results Ten potential targets of quercetin were identified, mainly enriched in autophagy-related pathways, and they demonstrated strong binding affinity in molecular docking analysis. qPCR results demonstrated that, compared with the human NPC line HNE1 transfected with the empty vector, the expression of LMP1 mRNA was significantly upregulated in both the HNE1-wild type (WT) and H101R - HNE1 cell lines (both P < 0.05). Compared with HNE1-WT cells, quercetin significantly inhibited proliferation and migration and promoted apoptosis in HNE1-H101R cells (all P < 0.05). Western blotting analysis showed no significant differences in LC3-Ⅱ/LC3-Ⅰ ratios or p62 protein levels between HNE1-WT and HNE1-H101R cells in the absence of quercetin (both P > 0.05). However, treatment with 100 μmol/L quercetin for 48 h significantly increased the LC3-Ⅱ/LC3-Ⅰ ratio and decreased p62 levels in HNE1-H101R cells (both P < 0.05). After HNE1-H101R cells were treated with 50 or 100 μmol/L quercetin together with the autophagy inhibitor chloroquine (2 μmol/L), the quercetin-induced inhibition of proliferation was reversed (both P < 0.05). Conclusion Quercetin may induce cell death in HNE1-H101R cells by promoting excessive autophagy.