This study evaluated the combined effect of fluoride varnish and Er:YAG laser on the permeability of eroded bovine root dentin. After initial erosive challenge followed by a remineralization period, the specimens were divided in two groups according to the treatment - fluoride varnish and non-fluoride varnish - and were subdivided according to the irradiation protocol: Er:YAG laser (100 mJ, 3 Hz, 12.8 J/cm2per pulse, non-contact and defocus mode) and non-irradiated. After a lead-in period, 7 volunteers wore a palatal device containing 4 specimens that were subjected to erosive challenges. At the first experimental phase, 4 volunteers used specimens treated with fluoride varnish and fluoride varnish+Er:YAG laser and 3 volunteers used specimens treated with non-fluoride varnish and non-fluoride varnish+Er:YAG laser. After a washout period, volunteers were crossed to treatments, characterizing a 2x2 crossover experiment. At the end of the experimental phase, the quantitative response variable was obtained by permeability analysis and the qualitative response by scanning electron microscopy (SEM). Two-way ANOVA and Tukey-Kramer's test revealed that specimens treated with fluoride varnish+Er:YAG laser showed the lowest permeability and a significant difference was found between this group and the others. When varnish (fluoride/non-fluoride) was applied in the absence of Er:YAG laser, higher permeability was found when compared to the laser-treated groups. SEM evaluations showed partially or completely obliterated dentinal tubules when specimens were treated with fluoride varnish+Er:YAG laser. It may be concluded that Er:YAG laser was able to control the permeability of eroded root dentin and the combination with fluoride varnish increased laser action.