Sunflower seed oil (SFO) (Helianthus annuus L.) is rich in oleic acid and unsaturated fatty acids and can potentially treat various ailments. To validate the ethno-therapeutic claims of the SFO in skin diseases, the incision wound healing activity was studied. The objectives of this study were to evaluate the chemical content and incision wound-healing activities of SFO. Twenty-seven adult male Wistar rats were used, and a 2 cm length and 2 mm depth incision was made on the back of the rat. The rats were administered SFO, povidone iodine (PI), or were left untreated as a control group. Histological evaluations were conducted on postoperative wound tissue biopsies on days 0, 5 and 10. The SFO compounds were assessed using GC/MS analysis, and a subsequent in silico investigation was conducted based on the GC/MS findings. The binding affinity of these compounds to TNF-α, VEGFR1, IGFR1 and TGF-β1 was evaluated. On day 10, sunflower seed oil topical application accelerated wound healing by decreasing wound area and increasing wound contraction compared to untreated and PI-treated wounds. Moreover, granulation tissue expanded more, and the epidermis fully recovered. Twenty-four active compounds were detected using GC/MS, and six are responsible for lipid metabolism regulation and anti-inflammatory activity. The in silico study showed that 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol (3ß,5Z,7E) had a high affinity of-8.5 kcal/mol for TNF-α. The conclusion of this study shows that SFO has incision wound healing activity in vivo and in silico by controlling the inflammatory phase and proangiogenic factors with a high affinity to bind to TNF-α in the skin tissue. © 2023, Romanian Society for Pharmaceutical Sciences. All rights reserved.