The exact function of eosinophils in cancer, particularly in oral squamous cell carcinoma (OSCC), has not yet been elucidated and the possible antitumor effect of these leukocytes is associated with the release of cytotoxic proteins, particularly eosinophil cationic protein (ECP). The aim of this study was to evaluate the effect of ECP on human OSCC lines and to provide novel insights into the role of eosinophils in these tumors. The viability of the SCC‑4 and SCC‑25 OSCC cell lines was assessed by colorimetric assay using 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT). The cells were plated into 96‑well plates in Dulbecco's modified Eagle's medium̸Ham's nutrient mixture F12 supplemented with 10% fetal bovine serum. After 24 h, the indicated concentration of ECP (0‑10 µM) was added to each sample. The plate was read using a microplate reader at a wavelength of 570 nm. The association between variables was estimated by linear regression analysis. There was a significant inverse association between ECP concentrations with SCC‑4 (β=0.16, P=0.019) and SCC‑25 cell viability (β=0.24, P=0.006). To the best of our knowledge, the present study was the first to investigate the effects of ECP on OSCCs and to demonstrate a significant inverse association between ECP concentrations with SCC‑4 and SCC‑25 cell viability.