Gram-negative bacterial pathogens are responsible for various infections. Over the past decade, these pathogens have acquired resistance to multiple antibiotics, and the multidrug-resistant (MDR) bacteria have rapidly spread globally, creating significant treatment challenges. Quaternized cellulose nanocrystals (CNCs) have promising antibacterial properties. We previously reported quaternized CNCs with ten-carbon (CNC-3) and sixteen-carbon (CNC-4) alkyl chains and an unmodified CNC (CNC-1). We found that CNC-4 exhibited a significant bactericidal effect against Staphylococcus aureus. In this study, we aim to evaluate the antibacterial properties of the quaternized CNCs against Gram-negative MDR clinical isolates of Acinetobacter baumannii (21 isolates), Klebsiella pneumoniae (18 isolates), and Escherichia coli (7 isolates), including each of their reference species. Agar diffusion, minimum bactericidal concentration (MBC), and bacterial killing pattern were conducted. The results showed that CNC-3 exhibited an MBC of 50 μg ml for 28% (13 out of 46 isolates) and 100 μg ml for 72% (33 out of 46 isolates), regardless of their antibiotic susceptibility. In comparison, CNC-4 exhibited an MBC of 100 μg ml for 28% (5 out of 18 K. pneumoniae), while all other isolates and the reference species exhibited an MBC of >100 μg ml. For CNC-1, the MBC was >100 μg ml for all tested isolates and the reference species. These results suggest that, unlike S. aureus, CNC-3 has a significantly higher and broader spectrum of bactericidal effects than CNC-4 against Gram-negative bacteria. This finding suggests that quaternized CNCs may be a potential antimicrobial agent for treating Gram-negative bacterial infections.