Electrospinning is a direct, continuous, and useful technique to prepare nanofiber by applying electrostatic forces. In this study, poly(lactic‐co‐glycolic acid)/poly(ethylene glycol) (PLGA/PEG) nanofiber mats were prepared, and electrospinning process was optimized to obtain appropriate fiber diameter and hydrophilicity for anti‐adhesion application. Optimization of applied voltage, PEG content, and feeding rate was investigated using response surface methodology. A total of 15 trials were designed to optimize the parameters. Fiber diameter was measured using scanning electron microscopy. Individual and interactive effects of the solution properties were determined. Moreover, the adequacy of the models was verified by validation experiments. For anti‐adhesion test, a nanofiber mat was produced based on the suggested optimum electrospinning conditions. Results showed that optimum fiber diameters were obtained using 7.5% PEG content, applied voltage of 19 kV, and flow rate of 3 mL/h. Experimental results were in good agreement with the predicted fiber diameters. Furthermore, a rat model of sidewall defect‐cecum abrasion was employed to investigate the efficacy of PEG/PLGA in preventing postoperative peritoneal adhesions. Hence, this study provides an overview on the fabrication of PLGA/PEG nanofibers with targeted diameter, which may be used in anti‐adhesion. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46282.