This study aimed to explore how the long-term use of midazolam (MDZ) influences the biochemistry and structural alterations in the salivary glands of adult rats, along with the function of their saliva. Twenty male Wistar rats were allocated in two groups (n = 10) and received daily intragastric administration of 0.1mg/kg MDZ solubilized in distilled water or only distilled water (control) for 30 days. Then, saliva, parotid, and submandibular glands were collected to perform histomorphometry analyses and biochemical assays. The groups were compared by using the Student t test (p < 0.05). MDZ decreased the reduced glutathione levels in the parotid and submandibular glands. In both glands, MDZ caused a reduction in the parenchymal area, an increase in the stromal area, and a reduction of in acinar area and size. In saliva, MDZ reduced trolox equivalent antioxidant capacity levels, and decreased the activity of salivary amylase. As a result of this study, we conclude that MDZ can trigger biochemical and morphological changes in the parotid and submandibular glands, as well as functional impairment and modulation of oxidative biochemistry in total saliva. With this, our pre-clinical study points to changes that could have an impact on biological processes such as tooth decay and food digestion.