BACKGROUND: A carbon dioxide (CO2) laser has been used to morphologically and chemically modify the dental enamel surface as well as to make it more resistant to demineralization. Despite a variety of experiments demonstrating the inhibitory effect of a CO2 laser in reduce enamel demineralization, little is known about the effect of surface irradiated on bacterial growth. Thus, this in vitro study was preformed to evaluate the biofilm formation on enamel previously irradiated with a CO2 laser (λ = 10.6 µM). METHODS: For this in vitro study, 96 specimens of bovine enamel were employed, which were divided into two groups (n = 48): 1) Control-non-irradiated surface and 2) Irradiated enamel surface. Biofilms were grown on the enamel specimens by one, three and five days under intermittent cariogenic condition in the irradiated and non-irradiated surface. In each assessment time, the biofilm were evaluated by dry weigh, counting the number of viable colonies and, in fifth day, were evaluated by polysaccharides analysis, quantitative real time Polymerase Chain Reaction (PCR) as well as by contact angle. In addition, the morphology of biofilms was characterized by fluorescence microscopy and field emission scanning electron microscopy (FESEM). Initially, the assumptions of equal variances and normal distribution of errors were conferred and the results are analyzed statistically by t-test and Mann Whitney test. RESULTS: The mean of log CFU/mL obtained for the one-day biofilm evaluation showed that there is statistical difference between the experimental groups. When biofilms were exposed to the CO2 laser, CFU/mL and CFU/dry weight in three day was reduced significantly compared with control group. The difference in the genes expression (Glucosyltransferases (gtfB) and Glucan-binding protein (gbpB)) and polysaccharides was not statically significant. Contact angle was increased relative to control when the surface was irradiated with the CO2 laser. Similar morphology was also visible with both treatments; however, the irradiated group revealed evidence of melting and fusion in the specimens. CONCLUSION: In conclusion, CO2 laser irradiation modifies the energy surface and disrupts the initial biofilm formation.