Breast cancer has a high prevalence among women which has high recurrences even after common therapies, so using nanofibers that contain natural compounds along with antineoplastic drugs locoregionally can be a novel supportive strategy. In this study, hot water extracted lentinan from Lentinula edodes (TMU340) was purified by ion-exchange chromatography. The effects of electrospun PVA/lentinan/docetaxel nanofibers were determined on HER3 expression in MCF-7 cell line. Lentinan concentration was determined at 0.243 mg/ml. The optimal electrospinning conditions were 15 kV, 15 cm, and 1 ml/h. The HER3 expression of cells treated with PVA/lentinan/docetaxel nanofibers obtained 0.52 in the most effective concentration at 72 h. Based on the results, PVA/lentinan/docetaxel nanofibers have shown appropriate synergistic effects on breast cancer. Therefore, the utilization of lentinan as a natural compound with a common chemical anticancer drug and nano-drug delivery system can be considered as a promising suggestion within the treatment of cancer.