Hypertrophic scarring of the skin is a cause of pain, disfigurement, and restricted mobility. Excessive TGF-β1 signalling leads to SMAD3 phosphorylation, which is implicated in hypertrophic scarring. In this study, we examined the mechanism of action of tomentosenol A, a small compound that we isolated from the propolis of the Australian stingless bee Tetragonula carbonaria. Cultured adult human dermal fibroblasts and HEK293 cells were stimulated with TGF-β1, with or without tomentosenol A, and were assessed for phosphorylation of SMADs 2/3 (Western blot, AlphaLISA assay), SMAD signalling (HEK293 cells expressing a SMAD3 reporter gene), and profibrotic gene transcription using RTqPCR for ACTA2 (smooth muscle α-actin), COL1A1 and COL3A (collagens), CCN2 (connective tissue growth factor) and FN1 (fibronectin). Protein expression was measured using ELISA (fibronectin) and visualised via confocal microscopy (smooth muscle α-actin). TGF-β1 increased SMAD3 phosphorylation by 44.3-fold above baseline levels, and this effect was inhibited by tomentosenol A in a concentration-dependent manner (IC50, 99.0 nM). TGF-β1 stimulated SMAD3 reporter gene expression and upregulated ACTA2, COL1A1, COL3A1, FN1 and CCN2 transcription; fibronectin protein expression; and smooth muscle α-actin filament formation in fibroblasts. These responses were inhibited by 6.25 μM tomentosenol A. These findings indicate that tomentosenol A inhibits TGF-β1/SMAD3 signalling and downstream profibrotic gene transcription and protein expression. As this pathway is implicated in hypertrophic scarring of the skin, tomentosenol A can be developed as a novel therapy for the management of scars caused by deep dermal injuries that are associated with surgery, trauma and burns.