The effect of Atropa belladonna L. (AB) aqueous extract on skin wound healing was studied in male Sprague-Dawley rats subjected to two parallel full-thickness skin incisions on the back. Specimens for histological evaluation were collected on days 2 and 5 whereas for biomechanical testing, they were collected on day 5. In the in vitro study, a different concentration of AB extract was used to test the differentiation of keratinocytes using a panel of selected antibodies, proliferation, and cell survival of 3T3 fibroblasts and human umbilical vein endothelial cells using the MTT-assay. Results of the in vivo experiments showed in AB-treated wounds a shortened process of inflammation and accelerated collagen formation, as well as significantly increased wound stiffness as compared with control tissues. The in vitro examination showed that control keratinocytes were cytokeratin 19 free, while samples exposed to the highest AB extract concentration expressed CK19. Moreover, all concentrations were stimulatory to human umbilical vein endothelial cell proliferation. In addition, only the AB extract at the lowest tested concentration increased fibroblast growth, but higher concentrations decreased cell survival. In conclusion, our results indicate that the AB water extract positively affects early phases of skin wound healing in rats. However, the in vitro results on the inverse relation between the concentration of the AB extract and its effects on cell proliferation may be important for future research.