The objective of this study was to evaluate the effects of application of different fluences and energies of laser in the 24-, 48-, and 72-h periods in fibroblasts originating from human skin (HFF-1). The cell used as a template for cell proliferation was HFF-1. For the photobiomodulation (PBM) application, a 660 nm laser with a power of 40 mW and energies of 0.84, 1.40, 5.88, and 6.72 J was used. Five experimental groups were studied: one control group (CG) with simulated PBM and four groups that received PBM in different doses. The changes observed after laser irradiation were evaluated by cell viability (trypan blue) and proliferation [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] tests. Intergroup comparisons were performed using two-way analysis of variance and the Tukey post hoc test (software GraphPad Prism 7.0). In the trypan blue test, the total number of cells was significantly different between the irradiated groups and the CG at all times studied. The total number of cells increased in laser group (LG)1 (0.84 J) and LG2 (1.40 J) and decreased in LG4 (6.72 J). The mitochondrial activity increased significantly in LG1 and LG2 at 48 and 72 h and decreased in LG3 (5.88 J) and LG4 (6.72 J) compared with CG. : The results indicate that the lower doses (0.45 and 0.75 J/cm) of PBM induce the highest mitochondrial activity and cellular viability.