The aim of this in vitro study was to evaluate the effects of a single dose application of two daily toothpastes on enamel exposed to acid attack. The research was conducted on human molars enamel fragments (n = 72). The two different toothpastes active ingredients were sodium fluoride (NaF) and stannous fluoride (SnF2). They were compared in protecting the surface of the enamel exposed to three acids: citric acid, lactic acid and hydrochloric acid. A spectrophotometer was used to measure the calcium ions and phosphate released in the solutions by the enamel specimens. Afterward, ionic concentrations were analyzed through the t-Student test, in order to estimate the significance level (p < 0.05) of the solubility differences obtained between the treatment and control groups. Finally, sample surfaces were analyzed with scanning electron microscopy and X-ray energy dispersive spectroscopy (SEM/EDX). The two analyzed toothpastes did not reveal any statistically significant variation in the release of calcium and phosphate (p > 0.05). Nevertheless, acid-resistant deposits were detected in samples treated with stannous fluoride and exposed to lactic acid, though the presence of tin ion deposits on samples treated with stannous fluoride was not shown. A single dose of a fluoride-based toothpaste before different acids attack, in simulated oral cavity conditions, did not show a significant preventive effect.