With the advent of nanotechnology has come an array of nanomaterials that can be used in biomedical applications. Inorganic nanoparticles have therapeutic potential due to their favourable properties, including small size, ease of synthesis, and high surface-to-volume ratio. However, functionalization with cationic polymers is required to enhance their stability and allow for therapeutic agent conjugation. Due to the potential associated toxicities of some nanoparticles and polymers, their therapeutic use has been halted. Hence, the study at hand evaluated the in vitro cytotoxicity of popular inorganic nanoparticles (NPs) (gold, silver, selenium, and palladium) and cationic polymers (poly-L-lysine, polyethyleneimine, and chitosan) in four mammalian cell lines. All NPs were chemically synthesized and characterized using UV-visible spectroscopy. NPs and polymers were morphologically and physiochemically characterized with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), which revealed that both NPs and polymers were within the nanometre size range. There was evidence to support the instability of uncoated NPs and the highly stable and cationic nature of the polymers tested. Cytotoxicity revealed that gold and palladium NPs were well tolerated across all cell lines, with silver NPs exhibiting high levels of cell death. Selenium NPs showed selective cell death in the cancer cell lines, confirming the anticancer properties of these NPs. All polymers were well tolerated in the non-cancer cells but exhibited slight toxicity levels in the cancer cells. Overall, this in vitro cytotoxicity study provides valuable information that can assist in the way forward for using these nanoparticles and polymers in nanomedicine for cancer therapy.