The aim of our study was to set up an approach for reliable biotreatability assessment of ionic liquids (ILs). As a case study, two different ILs were selected: pyridinium-based 1-butyl-3-methylpyridinium dicyanamide ([bmpyr][dca]) and imidazolium-based 1-butyl-3-methylimidazole tetrafluoroborate ([bmim][BF4]). Toxicity in aerobic conditions was determined by measurement of inhibition of oxygen consumption by activated sludge, while their biodegradability was calculated from measurements of oxygen consumption and dissolved organic carbon elimination. For their biotreatability in anaerobic conditions, the method with measurement of biogas production has been employed comparing flocculent and granular sludge. Both ILs were less toxic and more biodegradable in anaerobic conditions. IL [bmpyr][dca] was not toxic to granular sludge up to 742 mg L−1 and it even has been degraded at this concentration in the presence of easily degradable glucose. Flocculent sludge was completely inhibited at the lower concentration of 318 mg L−1, but it degraded by 44% at 106 mg L−1 in the presence of glucose, indicating the appearance of cometabolism. IL [bmim][BF4] was less toxic but also resistant to biodegradation in anaerobic conditions. It degraded via cometabolism 21% at 1,452 mg L−1. It has been concluded that any assessment of biotreatability of ILs should include parallel determination in aerobic and anaerobic conditions.