Chemoresistance and severe toxicities represent major drawbacks of chemotherapy. Natural extracts, including the essential oils of Pistacia lentiscus (PLEO), exhibit substantial anticancer and anti-inflammatory activities where different cancers are reported to dramatically recess following targeting with PLEO. PLEO has promising antimicrobial, anticancer, and anti-inflammatory properties. However, the therapeutic properties of PLEO are restricted by limited stability, bioavailability, and targeting ability. PLEO nanoformulation can maximize their physicochemical and therapeutic properties, overcoming their shortcomings. Hence, PLEO was extracted and its chemical composition was determined by GC-MS. PLEO and 5-Fluorouracil (5FU) were electrospun into poly-ε-caprolactone nanofibers (PCL-NFs), of 290.71 nm to 680.95 nm diameter, to investigate their anticancer and potential synergistic activities against triple-negative breast cancer cells (MDA-MB-231), human adenocarcinoma breast cancer cells (MCF-7), and human skin melanoma cell line (A375). The prepared nanofibers (NFs) showed enhanced thermal stability and remarkable physical integrity and tensile strength. Biodegradability studies showed prolonged stability over 42 days, supporting the NFs use as a localized therapy of breast tissues (postmastectomy) or melanoma. Release studies revealed sustainable release behaviors over 168 h, with higher released amounts of 5FU and PLEO at pH 5.4, indicating higher targeting abilities towards cancer tissues. NFs loaded with PLEO showed strong antioxidant properties. Finally, NFs loaded with either PLEO or 5FU depicted greater anticancer activities compared to free compounds. The highest anticancer activities were observed with NFs co-loaded with PLEO and 5FU. The developed 5FU-PLEO-PCL-NFs hold potential as a local treatment of breast cancer tissues (post-mastectomy) and melanoma to minimize their  possible recurrence.