Fibroblasts are actively involved in the formation of granulation tissue and/or tumor stroma. These cells possess the potential to differentiate into myofibroblasts acquiring a highly contractile phenotype characterized by the expression of α-smooth muscle actin (SMA). Considering TGF-β1 as the main inducer of myofibroblast differentiation and horse chestnut extract (HCE) as an effective modulator of the wound healing, we have new evidence to demonstrate canonical TGF-β1/SMAD and non-canonical/non-SMAD signaling in normal fibroblasts, isolated from healthy human skin (human dermal fibroblasts - HDFs), and their malignant counterparts (CAFs) isolated from basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) using western blot and immunofluorescence. Our study revealed that HCE stimulated the deposition of fibronectin by BCC fibroblasts (BCCFs), an effect not seen in other studied fibroblasts. Moreover, HCE in combination with TGF-β1 showed a synergic effect on the presence of polymerized SMA-stress fibers, particularly visible in CAFs. Interestingly, the TGF-β1 exposure led to activation of the canonical SMAD signaling in HDFs and BCCFs, whereas treatment of SCC fibroblasts (SCCFs) resulted in activation of the non-canonical AKT and/or ERK1/2 signaling. In conclusion, we observed specific differences in signaling between HDFs and CAFs that should be considered when developing new therapeutic approaches targeting wound/tumor microenvironments.