The present study for the first time demonstrated the successful fabrication of camptothecin (CPT) loaded poly(ε-caprolactone) (PCL) nanofibrous mats through one-step sol–gel electrospinning process. Encapsulation of CPT by PCL was done to modulate the CPT release and to improve the stability and anticancer activity of the released drug. The physicochemical characterization of as-spun nanofibers was carried out by field emission scanning electron microscopy (FE-SEM), differential scanning calorimetry (DSC), UV–visible spectroscopy (UV–vis), liquid chromatography mass spectrometry (LC–MS), thermogravimetry (TGA), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) pattern. The medicated nanofibers were evaluated for their cytotoxicity on C2C12 cells using cell counting kit-8 assay. The in vitro studies indicated a slow and prolonged release over a period of 144h with mild initial burst. XRD, DSC and TGA studies confirmed the polymer formation and interactions among the anticancer drug CPT and PCL matrix. Evidently, the chemical integrity of the as-loaded CPT was sustained after electrospinning as the peaks corresponding to CPT were observed in the LC–MS spectrum of drug loaded nanofibrous mat. The cytotoxicity results showed that the CPT loaded PCL nanofibers were more cytotoxic than the free drug during 72h incubation period. To employ a drug carrier system providing sustained release for long time duration might be a suitable strategy to maximize the drug content within unhealthy cells. Thus our results emphasize the potential of CPT loaded PCL nanofibers as a chemotherapeutic agent for delivery of CPT in order to treat different malignancies.