This study investigated the impact of subcritical water extraction parameters on the polyphenols and flavonoids content in extracts made of the bark of walnut (Juglans regia L.), as well as antioxidant activity measured using the DPPH and FRAP methods. The total polyphenols (TPC) and flavonoid (TFC) were determined using the spectrophotometric (UV-Vis) method. The response surface methodology (RSM) was employed to optimize the yield of bioactive compounds. On the basis of developed model, the highest polyphenols (192.2 mg (GAE)/100 g (dry mass)) (GAE—gallic acid equivalent), flavonoids (88.3 mg(QE)/100 g (dry mass)) (QE—quercetin equivalent) contents and antioxidant activity (21.3 × 10−6 MTE/1 g (dry mass)) (TE-Trolox equivalent) were obtained under the following extraction conditions: temperature 131.6 °C, raw material fraction size 0.9 mm and process time 10 min. The greatest impact on the extraction yield was observed in the case of the temperature, whereas the raw material fraction size and the process time had a secondary impact. Obtained results indicate that a smaller particle size and a shorter time may improve the process; however, a reduction in particle size can be challenging for a pressure cell filtration system and consequently increase the cost of the raw material pretreatment process. With the reduction in the process time, a lower cost of extract production can be achieved. This study confirmed the importance of a proper selection of extraction parameters to obtain the demanded extract composition.