The objective of the study was to evaluate the anticancer activity of water‐soluble peptide (WSP) extracts of buffalo and cow milk Cheddar cheeses during their ripening. The WSP extracts were assessed for their effects on cell viability, cell cycle arrest and apoptosis induction using a lung cancer (H‐1299) cell line. Cheese extracts taken after 120 and 150 ripening days showed marked growth inhibition of the cancer cells, in a dose‐dependent manner. During cell cycle analysis, a significant increase in cells population at G₂/M phase was observed in the lung cancer cell line at a rate of 400 μg/mL and 500 μg/mL. In addition, the extracts also induced extensive apoptosis in the lung cancer cell line.