This study aims to examine in detail for the first time the cytotoxic profile of twelve choline chloride-based deep eutectic solvents (NADES) against HT-29, Caco-2, MCF-7, and MRC-5 cell lines. All NADES systems were synthesized by microwave synthesis using choline chloride as a hydrogen bond acceptor (HBA) and selected sugars, alcohols, organic acids, and urea as hydrogen bond donors (HBD) with the addition of 20% water (w/w) to all systems. It was observed that the cytotoxic effect predominantly depended on the structure of HBD. Acidic systems, where HBDs were organic acids showed the highest cytotoxic effects in all investigated cell lines. The cytotoxicity depended mostly on the concentration of the NADES system in the cell medium as well as on the chemical constitution of the investigated systems. The highest cytotoxic effects showed acidic systems, especially to the HT-29 cell line. The EC value for the citric acid-based system was 3.91 mg mL for the HT-29 cell line which was the most vulnerable to acidic NADES systems.