Deep eutectic solvents (DESs) were described at the beginning of 21st century and they consist of a mixture of two or more solid components, which gives rise to a lower melting point compared to the starting materials. Over the years, DESs have proved to be a promising alternative to traditional organic solvents and ionic liquids (ILs) due to their low volatility, low inflammability, easy preparation, and usually low cost of compounds used in their preparation. All these properties encouraged researchers to use them in diverse fields and applications e.g., as extractants for biomolecules and solvents in pharmaceutical and cosmetic industries. Nevertheless, despite undeniable potential of DESs, there is still controversy about their toxicity. Besides the low number of studies on this topic, there are also some contradicting reports on biocompatibility of these solvents. Such misleading reports could be mainly attributed to the lack of well design standard protocol for DESs toxicity determination or the use of out-off-purpose methodology. Thus, to better apply DESs in green and sustainable chemistry, more studies on their impact on organisms at different trophic levels and the use of proper techniques are required. This review focuses on DESs toxicity towards microorganisms and is divided into three parts: The first part provides a brief general introduction to DESs, the second part discusses the methodologies used for assessment of DESs microbial toxicity and the obtained results, and finally in the third part the critical evaluation of the methods is provided, as well as suggestions and guidelines for future research.