Biodegradable polyethylene oxide (PEO)/chitosan (CS)/poly(epsilon-caprolactone) (PCL)/olive oil composite nanofibers were fabricated by electrospinning process. The prepared nanofibers were characterized using SEM and FTIR analysis. A response surface methodology based on Box-Behnken design (BBD) was used to predict the average diameter of electrospun nanofibers based on electrospinning parameters including voltage, flow rate and tip-collector distance. The optimum experimental average diameter of electrospun nanofibers was found to be 86 nm which was in good agreement with the predicted value by the BBD analysis (88 nm). In vitro release of olive oil incorporated PEO/CS/PCL/olive oil nanofibers demonstrated a rapid release of olive oil during the first 3 h which enhanced gradually afterwards. Good attachment, spreading, cell proliferation, as well as nontoxic behavior of PEO/CS/PCL/olive oil nanofibrous scaffolds on HER fibroblast cells were proved by cytotoxicity studies. Furthermore, the high antibacterial activity of PEO/CS/PCL/olive oil composite nanofibers against Gram-negative bacteria E. coli and Gram-positive S. aureus was observed. This study suggests that the prepared PEO/CS/PCL/olive oil composite nanofibrous scaffolds could be used as an ideal patch for wound dressing applications.