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Mouthwashes (MWs) are liquid preparation, commonly used for oral care and applied on teeth and mucosa of the oral cavity and pharynx. MWs exert antiseptic, astringent, and sedative effects. The aim of the current study is to investigate the effect of daily use of alcohol-containing mouthwashes on the tongue, surface of cementum and enamel in rats. Four groups (10 rats/group) of male Sprague–Dawley rats were treated by local application of MWs by pledgets for 45 min for 41 days as follows: The control group treated with artificial saliva; the group treated with Lacalut® (MW1); the group treated with Listerine® (MW2); the group treated with Tricare® (MW3). At the beginning, mandibular of first molar was extracted from each animal and soaked into artificial saliva or in MW for an in vitro study. At the end of the treatment, tongue, and extracted mandibular first molar from each group were collected for histological examinations and Transmission Electron Microscopes (TEM). Alcohol-containing mouthwashes showed sever histological changes and keratosis of tongue. Scanning electron microscope revealed that MW3 induced sever distortion of dorsal surface of the tongue and a distinguished harmful effect on enamel and cementum surface. It could be concluded that daily use of alcohol-containing mouthwashes induced harmful effect on dorsal surface of tongue, dental erosion on enamel and cementum surfaces.