Introduction: 5-Fluorouracil (5-FU) is a common chemotherapeutic drug for treatment of oral squamous cell carcinoma. However, its toxicity to normal tissues has limited its role as an effective cancer therapy. Aim: This research aimed to investigate the effect of combining 5-FU with honokiol (HNK) on enhancing the anticancer activity of 5-FU without increasing its toxicity. Material and methods: HNK was used in combination with 5-FU for treatment of tongue carcinoma induced chemically by 4-nitroquinoline 1 oxide (4-NQO) in albino rats. Rats were divided into five groups (10 animals each) including control, 4-NQO, 5-FU, HNK, and combined 5-FU with HNK. Serial sections of the tongues from all animals were examined microscopically and real-time PCR quantification of P53 gene expression was also assessed. Results: The results showed that treatment with both 5-FU + HNK had significantly regressed the number and size of malignant lesions seen in the carcinogen-only group. Data analysis revealed a statistically significant decrease in the expression levels of mutant P53 in the combined treatment group compared to control groups. Conclusion: This study reported that HNK and 5-FU had a synergistic cytotoxic effect on this model, without notable intensification of the side effects associated with 5-FU treatment.