Oral mucositis (OM) is a common inflammatory complication among cancerous patients as an adverse effect of chemotherapy and radiotherapy. The aim of this study is to evaluate the healing effects of hydroalcoholic extract of Psidium Guajava leaf on oral induced mucositis induced by 5-fluorouracil using histopathologic and tissue antioxidative markers assessment in male dark brown rats. In a prospective randomized double blind animal study, OM was induced in 64 male dark brown rats that allocated in 4 groups by 5-FU (60 mg/kg) on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle on once daily on days 3 and 4. Starting from day 12, gel base, topical form and 600 mg/kg dietry form of hydroalcoholic extract of Psidium Guajava leaf were administered per day. Pouch histopathology score, superoxide dismutase, glutathione peroxidase, total antioxidant capacity were evaluated on day 14 and 18. DPPH scavenging activity and total phenolic content also were measured. Histopathology scores of mucositis were lower in the systemic and topical treatment groups than the gel base and control groups (P<0.05). Higher activities of SOD, GPX and TAC were detected in the topical and systemic treatment groups in comparison to the others (P<0.05). The extract was rich in total phenolic content as antioxidant. The use of extract of Psidium Guajava leave may be associated with reduced intensity of OM, increased concentration of SOD, GPX and TAC on induced OM in dark brown rats undergoing 5-FU consumption. This may be due to appropriate antioxidant activity and phenolic contents.