Ionic liquids (ILs) are being considered as greener alternatives to conventional organic solvents. Recent applications of ILs in the dissolution and stability of biomolecules have led to increasing demand for biocompatible ILs. In the present work, we report the synthesis and toxicity study of tetramethylguanidinium [TMG] cation based ILs with benzoate, salicylate, lactate, dihydrogen phosphate, nitrate, formate, acetate, propanoate, butanoate and valerate anions. The results obtained from toxicological evaluation with normal HEK293 and cancerous DLD-1 cell cultures show that all studied ILs are non-toxic. Subsequent biodegradability tests with bacterial cultures Pseudomonas putida and Bacillus subtilis and a closed bottle test show that these ILs are also biodegradable. Our study suggests that the side-chain of the IL anions and anion-cation combination can influence the biocompatibility of the ILs immensely. © 2017 The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.