In nanomedicine, gold nanoparticles (GNPs) are based on metal-based nanomaterials and have attracted considerable attention from researchers for their use in drug delivery, including wound healing. This study examined the use of Gum tragacanth (GT) for producing gold nanoparticles (GNPs) through the green synthesis method (GT-GNPs). The antibacterial activity of GT-GNPs against pathogenic bacterial strains, as well as assess the wound healing potential of GT-GNPs combined with photobiomodulation (PBM) on normal and diabetic wound models (in vitro). In physicochemical characterization results, we found that the synthesized nanoparticles are 10-20 nm with an average of 12 ± 2 nm in size, and smooth surface and confirmed the presence of GT on the GT-GNPs. It also possessed optimal antibacterial activity and was confirmed through flow cytometry studies. Furthermore, we examined the combined effect of GT-GNPs and PBM against normal and diabetic wound models in WS1 (Humal Fibroblast cells), including cell morphology, percentage of wound closure, nuclear, and filamentous (F)-actin morphology. The combined effects of GT-GNPs and PBM effectively achieved progressive wound closure. In summary, this study has demonstrated that treating GT-GNPs has no adverse effects on both normal and diabetic wound healing processes.