Chronic wounds pose significant challenges in healthcare due to impaired healing mechanisms. Fibroblast cells play a crucial role in wound healing by orchestrating proliferation and migration. This study aimed to assess fibroblast cells derived from chronic wounds and explore the impact of low-level laser therapy (LLLT) on their growth and migration. Dermal samples from chronic pressure ulcers and diabetic ulcers were obtained from 20 patients at three sites. Fibroblasts from wound base, margins, and adjacent healing skin were isolated and characterized. Proliferation and migration capabilities of these cells were evaluated. LLLT was applied at various energy levels (2.5, 3, 3.5, 4, and 5 J/cm²) to assess its effect on cell count. Fibroblasts from chronic wounds exhibited slower proliferation and migration rates compared to normal dermal fibroblasts. Notably, LLLT intervention at different energy levels led to a significant increase in cell count, with the most pronounced effect observed at 3 J/cm². LLLT at an energy level of 3 J/cm² demonstrated a notable enhancement in fibroblast migration. These findings underscore the potential of LLLT as a therapeutic approach for chronic wounds, offering insights into its efficacy in augmenting fibroblast functions crucial for wound healing.