Polyethylene glycol (PEG) derivatives have been widely used in bio-related research. However, PEG oligomers (with different molecular weights) or PEG based monomers (with different chain end groups) actually have different chemical and physical properties, which might lead to potential toxicity. In this work, the cytotoxicity of a series of PEG derivatives (oligomers and monomers) has been measured using human cervical cancer cells (HeLa) and a cell line of fibroblasts derived from mice (L929) as model cells. Most of the PEG oligomers are safe to both types of cells except triethylene glycol (TEG), which is toxic at high concentrations to L929 cells. On the other hand, PEG-based monomers including poly(ethylene glycol) methyl ether acrylate (mPEGA) and poly(ethylene glycol) methyl ether methacrylate (mPEGMA) showed obvious cytotoxicity. Subsequently, those toxic PEG derivatives have been studied to reveal the different mechanisms of their toxicity. This current research evaluated the cytotoxicity of PEG derivatives and pointed out the potential hazard of ‘safe’ biomaterials, which might offer a useful reference for people to use the PEG derivatives in future biomedical research.