Some species of the Berberidaceae family and natural biopolymer chitosan have good effects on wound healing. In this research, a new formulation of chitosan hydrogel containing alcoholic root extract of Berberis integerrima (Chi-BIE) was designed and evaluated for the treatment of full-thickness skin wounds in an animal model. Alcoholic extract of Berberis integerrima root (BIE) was prepared, and its total amount of berberine was determined using UV/visible spectroscopy. Chitosan gel containing 10 and 20% BIE was prepared, and their extract release profile was assayed. The antimicrobial activity of Chi-BIE was investigated by the minimum inhibitory concentration assay. Finally, the efficiency of Chi-BIE formulations on the full-thickness wound healing was physically and histologically evaluated in an animal model. BIE was successfully loaded into the chitosan hydrogel and showed a burst release of extract during the first 4 h (60%) followed by a sustained release. Formulation of BIE in chitosan hydrogel could increase the antimicrobial activity of chitosan and BIE alone against Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilius, Escherichia coli, and Candida albicans. Chi-20% BIE indicated more antimicrobial activity compared to Chi-10%BIE. Formulation of BIE in chitosan hydrogel could increase the wound healing effects of chitosan and BIE alone. Chi-20% BIE had a faster restorative effect than other treatment groups due to more formation of epithelial tissue and faster maturation of bud tissue. Based on the results of this study, the chitosan hydrogel containing BIE is proposed as a suitable dressing for wound-healing.