The aim of this experimental study was to investigate the cytotoxic effects of intranasal midazolam on nasal mucosal tissue in rats. Forty healthy rats were randomly divided into 5 groups. Group 1 (n = 8) was the control group, group 2 (n = 8) received intranasal saline, group 3 (n = 8) received intranasal midazolam, group 4 (n = 8) received intraperitoneal saline, and group 5 received intraperitoneal midazolam (n = 8). Midazolam and saline were administered via intraperitoneal and intranasal routes at doses of 200mg/kg. Nasal septal mucosal stripe tissues were removed at the 6th hour. All materials were evaluated according to Ki67 and p53 staining to evaluate proliferation and apoptosis, respectively, and hemotoxylin and eosin staining was performed for histopathology evaluation. Ki67 values and inflammation in group 3 were statistically higher compared to group 1, group 2, and group 4. P53 values in group 3 were statistically higher compared to group 1. Assessment of subepithelial edema between group 3 and the other groups revealed no statistically significant differences. Assessment of cilia loss between group 3 and group 1, group 2, and group 4 revealed no statistically significant difference. The evaluation of goblet cell loss between group 3 and group 1 revealed a statistically significant difference. Intranasal midazolam had adverse effects on nasal mucosa. However, intranasal midazolam is as safe as systemic midazolam administration with respect to nasal mucosa.