Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cold plasma-stimulated medium (PSM) have been shown to exhibit tumor-selective cytotoxicity and have emerged as promising new tools for cancer treatment. However, to date, at least to the best of our knowledge, no data are available as to which substance is more potent in killing cancer cells. Thus, in this study, we systematically compared their abilities to kill human malignant cells from different origins. We found that PSM dose-dependently killed TRAIL-resistant melanoma, osteosarcoma and neuroblastoma cells. Moreover, PSM had little cytotoxicity toward osteoblasts. PSM was more potent than TRAIL in inducing caspase-3/7 activation, mitochondrial network aberration and caspase-independent cell death. We also found that PSM was more potent in inducing plasma membrane depolarization (PMD) and disrupting endoplasmic-mitochondrial Ca2+ homeostasis. Moreover, persistent PMD was caused by different membrane-depolarizing agents; the use of the anti-type II diabetes drug, glibenclamide, alone caused mitochondrial fragmentation and enhanced TRAIL-induced Ca2+ modulation, mitochondrial network abnormalities and caspase-independent cell killing. These results demonstrate that PSM has a therapeutic advantage over TRAIL owing to its greater capacity to evoke caspase-independent cell death via mitochondrial network aberration by disrupting membrane potential and Ca2+ homeostasis. These findings may provide a strong rationale for developing PSM as a novel approach for the treatment of TRAIL-resistant malignant cells.