Objective: To analyze the evolution of the thickness, bacterial vitality, covering grade, and the structure after 2 and 4 days of aging in "non-disturbed" plaque-like biofilm (PL-biofilm). Method and Materials: Twenty healthy volunteers wore a specific appliance. After 2 days half of the samples were removed from the appliance. Posteriorly, after bacterial vital staining, samples were analyzed using a confocal laser scanning microscope. In the first volunteer, one of the disks was analyzed using a scanning electronic microscope. The same process was realized on the remaining disks after 4 days. Results: The thicknesses of the PL-biofilm after 2 and 4 days were not significantly different. The bacterial vitality changed significantly from 72.50 ± 15.50% to 57.54 ± 15.66% over time, which was in contrast to the covering grade (53.08 ± 18.03% and 70.74 ± 19.11%). The structure changed from an irregular surface and compact deepest layer with a high predominance of the coccus shape to a complex structure with voids in the deepest layer and a great proportion of bacillus-shaped bacteria. Conclusion: The PL-biofilm thickness remained practically constant, decreasing the bacterial vitality and increasing the covering grade over time. Regarding the structure, differences were principally bacterial disposition in the surface and bacterial shape. Clinically, the findings show that new control strategies for combating the oral biofilm should be focused on inhibiting bacterial adhesion to tooth surfaces, which would reduce biofilm formation. Keywords: bacterial vitality, covering grade, laser scanning confocal microscopy, plaque-like biofilm, scanning electron microscopy, structure, thickness