A new class of betaine-based ionic liquids (ILs) have been synthesized. Betaine is transformed into betaine amides with various anions. These amides were prepared at first through a classical four-step method; the synthesis was then improved by reducing the number of steps using a one-pot strategy for amidation. The final yields were good to high, and the various structures were fully characterized to valorize them in many domains such as catalysis, extraction, or dissolution of biopolymers. Next, to determine their ecotoxicity, a hemocyte-based bioassay was used. This new method to evaluate the ecotoxicity of ILs was also applied to other ILs [tetrabutyl-ammonium (TBA), tetrabutyl-phosphonium (TBP), and betaine- and cholinium-based ILs] to prove its efficiency and its complementarity toward other ecotoxicological assessment methods. TBA- and TBP-based ILs induced limited effects on zebra mussel hemocytes, with TBP-ILs proving to be generally less toxic than TBA-based ones. On the contrary, betaine- and cholinium-based IL exposure led to more acute toxicity with a noticeable effect associated with the cation carbon chain length. Overall results also showed differential toxicity of ILs according to the nature of the anion in the assemblage, with lactate being the less biologically reactive anion. The results of this study thus reveal the importance of the nature of both cations and anions which constitute the IL structures and can strongly, sometimes synergistically, influence the IL toxicity. The possible interactions between the cationic and anionic parts reveal the complexity of IL toxicity prediction, thereby arousing interest in developing innovative strategies for the risk assessment of these molecular assemblages. In this context, the proposed hemocyte-based bioassay proved its efficiency and sensitivity in scoring and ranking IL toxicity according to their effects in the immunocompetent cells of an environmentally sentinel bivalve. This alternative bioassay could represent a complementary tool to more traditional and standard bioassays to complete the toxicity screening of substances such as ILs or new chemicals synthesized under green chemistry principles and notably the limitation of environmental toxicity by investigating the acute and functional effects on a wild and relevant freshwater invertebrate species.