Medication repurposing is one of the effective strategies in approving drugs for new therapeutic indications. Clarithromycin is a well-known macrolide antibiotic that is commonly used for the treatment of a wide variety of bacterial infections. However, extensive preclinical and clinical studies have recently revealed the efficacy of clarithromycin in treating various tumors in combination with conventional therapy. In this study, we formulated a clarithromycin nanoemulsion using pumpkin seed oil as a natural nano-carrier to study the possible cytotoxic effects against colorectal (HCT 116), breast (MCF-7), and liver cancer (HepG2) in vitro. The formulated nanoemulsion was characterized for droplet size distribution, surface charge, drug loading and in vitro drug release. The droplet size of clarithromycin nanoemulsion was 360.6±42.5 nm and zeta potential was −31.6± 2.8 mV. Incorporating the lipophilic drug, clarithromycin, within nanoemulsion significantly heightened in vitro drug release, compared to plain drug. The anticancer properties of the nanoemulsion formulation were examined using sulforhodamine B (SRB) assay as well as cell cycle and apoptosis analyses. Clarithromycin-loaded pumpkin seed oil-based nanoemulsion remarkably diminished the viability of all tested cell lines, compared to either plain clarithromycin or plank pumpkin seed oil nanoemulsion. In addition, incorporating clarithromycin within pumpkin seed oil-based nanoemulsion synergistically augmented the cytotoxic efficacy of clarithromycin against various cancer cell lines via advocating considerable cell cycle arrest with subsequent elicitation of potent apoptotic response. These results support the potential use of clarithromycin in cancer therapy, and merit future translational research.