<p>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 <i>Staphylococcus aureus</i>. In this study, we aim to evaluate the antibacterial properties of the quaternized CNCs against Gram-negative MDR clinical isolates of <i>Acinetobacter baumannii</i> (21 isolates), <i>Klebsiella pneumoniae</i> (18 isolates), and <i>Escherichia coli</i> (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⁻¹ for 28% (13 out of 46 isolates) and 100 μg ml⁻¹ for 72% (33 out of 46 isolates), regardless of their antibiotic susceptibility. In comparison, CNC-4 exhibited an MBC of 100 μg ml⁻¹ for 28% (5 out of 18 <i>K. pneumoniae</i>), while all other isolates and the reference species exhibited an MBC of &gt;100 μg ml⁻¹. For CNC-1, the MBC was &gt;100 μg ml⁻¹ for all tested isolates and the reference species. These results suggest that, unlike <i>S. aureus</i>, 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.</p>