Phototherapy plays a key role in wound healing and tissue regeneration. The use of lasers has the potential to become an effective and minimally invasive treatment in periodontal and peri-implant disease. The aim of this study was to evaluate the influence of three laser wavelengths with the combination of parameters such as power density and energy density on human gingival fibroblasts (hGFs) in vitro culture. Isolated cells were seeded in 96-well plates with culture medium (DMEM, Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum (FBS). After 24 h cells were irradiated (1064, 980 and 635 nm, various energy density value). After 24, 48 and 72 h, cells were evaluated for viability. Data were analyzed by ANOVA followed by Tukey’s HSD test. We found the best outcomes for hGFs irradiated with laser 1064 nm for all combinations of power output (50/400/1000 mW) and energy dose (3/25/64 J/cm2) after 48 h and 72 h compared with control group. Cell viability increase ranged from 0.6× (3 J/cm2, 50 mW) to 1.3× (64 J/cm2, 1000 mW). Our findings indicate that the appropriate use of low-level laser irradiation (LLLI) can increase the proliferation rate of cultured cells. The use of LLLI can be extremely useful in tissue engineering and regenerative medicine.