Epidermal growth factor receptor (EGFR) is a key molecule in the pathophysiology of oesophageal squamous cell carcinoma (OSCC). However, EGFR-targeted agents such as anti-EGFR antibody or tyrosine kinase inhibitors for OSCC have not demonstrated any clinical benefits. Recently, a novel chemotherapeutic agent, EGFR(2R)-lytic hybrid peptide, a composite of EGFR-binding peptide and lytic peptide fragments, has been shown to exhibit a potent anti-tumour effect against cancers that express high EGFR levels. In this study, we investigated the validity of employing EGFR(2R)-lytic hybrid peptide against OSCC cells both in vitro and in vivo. Additionally, the toxicity of this peptide was assessed in mice. We found high EGFR expression levels on the cell surface of OSCC cells, and the EGFR-binding peptide fragment showed high affinity for OSCC cells. A potent cytotoxic effect was induced within 30 minutes by the exposure of OSCC cells to EGFR(2R)-lytic hybrid peptide. Furthermore, EGFR(2R)-lytic hybrid peptide markedly suppressed the tumour growth of OSCC cells in a xenograft model. Moreover, it did not cause any identifiable adverse effects in mice. Taken together, EGFR(2R)-lytic hybrid peptide was shown to be a valid therapeutic agent against OSCC, providing a crucial rationale regarding novel EGFR-targeted therapies against OSCC.