The need for effective and safe therapies for cancer is growing as aging is modifying its epidemiology. Cold atmospheric plasma (CAP) has gained attention as a potential anti-tumor therapy. CAP is a gas with enough energy to ionize a significant fraction of its constituent particles, forming equal numbers of positive ions and electrons. Timely-resolved output voltage measurement, emission spectroscopy, and quantification of reactive species (RS) in plasma-activated media (PAM) were performed to characterize the physical and chemical properties of plasma. To assess the cytotoxicity of cold atmospheric plasma in human tumors, different cell lines were cultured, plated, and exposed to CAP, followed by MTT and SRB colorimetric assays 24 h later. Human fibroblasts, phenotypically normal cells, were processed similarly. Plasma cytotoxicity was higher in cells of breast cancer, urinary bladder cancer, osteosarcoma, lung cancer, melanoma, and endometrial cancer. Cytotoxicity was time-dependent and possibly related to the increased production of hydrogen peroxide in the exposed medium. Sixty seconds of CAP exposure renders selective effects, preserving the viability of fibroblast cells. These results point to the importance of conducting further studies of the therapy with plasma.