Abstract Numerous studies have reported cold atmospheric plasma cytotoxic activities in various cancer cell lines, either by direct exposure to non-thermal plasma or indirectly by activating a medium (plasma-activated medium, PAM) prior to cell treatment. We suggested the use of in vitro 3D tumor model spheroids to determine the potential of PAM for cancer therapy at the tissue scale, especially in human tumor tissue. This work aimed to better understand the effect of PAM on human colorectal tumor spheroids by describing the in vitro-induced-cell death kinetics and associated mechanisms to further improve its therapeutic potential. Tumor spheroid growth was delayed depending on contact time with PAM. Medium osmolarity was increased by activation with low temperature Helium plasma jet but it did not fully explain the observed growth delay. PAM impaired tumor cell viability through intracellular ATP depletion, leading within hours to both cell apoptosis and necrosis as well as mitochondrial oxidative stress. When successive treatments were spaced over time, cumulative effects on the growth delay of spheroids were observed. Taken together, these results demonstrated that plasma-activated liquids may represent a novel and efficient therapeutic method for the treatment of tumors, especially when successive treatments are applied.