Reactive species (RS) are the key factors of plasma‐activated solutions (PAS) against cancer. However, the specific contribution of each RS in inducing cancer cell death remain unclear. Therefore, this study aimed to evaluate the anticancer potential and mechanism of plasma‐activated medium (PAM) and plasma‐activated phosphate buffer saline (PA‐PBS) in vitro using colorectal cancer cells HCT116. Results showed that PAM and PA‐PBS effectively inhibited the growth and proliferation of HCT116 cells, with PA‐PBS exhibiting greater potency. H2O2, intracellular reactive oxygen species, and pH (H+) were essential mediators of cell death, and ONOO− exhibited cytotoxicity only after reaching a specific concentration. Additionly, PAS induced cell death through the exogenous apoptotic pathway.