NO (nitric oxide) molecule is produced by various mammalian cell types and plays a significant role in inflammation, infection and wound healing processes. Recently, gNO (gaseous nitric oxide) therapy has been utilized for its potential clinical application as an antimicrobial agent, with special focus on skin infection. In a previous study, we demonstrated that 200 ppm gNO, 8 h/day for three consecutive days significantly reduced the number of bacteria in dermal wounds without compromising the viability and function of skin cells. To increase the feasibility and ease of its clinical use, we propose that different doses of gNO (5 to 10 K ppm) for 8 h and as short as 10 min be used, respectively. To achieve this, we set up in vitro experiments and asked whether (i) different doses of gNO have any toxic effect on immune cells and (ii) gNO has any modulating effect on key ECM (extracellular matrix) components in fibroblasts. To further investigate the effect of gNO, expression of more than 100 key ECM genes have been examined using gene array in human fibroblasts. As immune cells play an important role in wound healing, the effect of gNO on proliferation and viability of human and mouse lymphocytes was also examined. The findings showed that, the 5, 25, 75 and 200 ppm of gNO for 8 h slightly increased the expression of Col 5A3 (collagen type V alpha 3), and gNO at 5 ppm decreased the expression of MMP-1 (matrix metalloproteinase 1), while exposure of fibroblast to 10 K ppm of gNO for 10 min does not show any significant changes in ECM genes. Exposure to gNO resulted in inhibition of lymphocyte proliferation without affecting the cell viability. Taken together, our findings show that skin could be treated with gNO without compromising the role of ECM and immune cells in low concentrations with long time exposure or high concentrations for a shorter exposure time.