Introduction: Fibroblasts are the most important cells in wound healing. Chlorhexidine is the most widely used and investigated chemical plaque control agent after periodontal surgeries and Low-level laser therapy (LLLT), or photo biomodulation (PBM), refers to using photons to modulate biological activity. Since literature have shown promising results on fibroblasts viability following LASER irradiation, we aimed to evaluate and compare the effect of chlorhexidine mouthwash and LASER on survival of human gingival fibroblasts. Material and Methods: The experimental and control groups are as follows: Group I - Control, Group II - Fibroblasts treated with Chlorhexidine (Peridex), Group III - Fibroblasts irradiated with diode LASER. The cells in group II were treated with Chlorhexidine (Peridex-0.12%) whereas the cells in group III were irradiated diode LASER 5 seconds and cell viability was assessed using MTT Assay. Statistical Analysis: Comparison between all three groups was done by Kruskall Wallis ANOVA Inter group comparison (>2 groups) was done using Kruskall Wallis ANOVA followed by pair wise comparison using Mann Whitney U test. Results: No change in the viability of cells in the control group, whereas the viability decreased to 50.4 % in group 2, which was statistically significant when compared with the control group (p<0.01) and increased to 101.86 % in grp 3, but statistically insignificant when compared to control group (p>0.01). Also, there was a statistically significant difference observed between group 2 and group 3 (p<0.01). Conclusion: Chlorhexidine treatment to fibroblasts resulted in the decreased viability of the fibroblasts, and LASER irradiated cells, neither showed a significant reduction nor a significant increase in viability of fibroblasts. And when the two test groups, chlorhexidine treated and LASER irradiated groups, were compared there was a statistically significant difference observed.