Background: Multidrug resistance caused by Gram-negative pathogens is a significant global health concern. The increased resistance of these pathogens to commonly prescribed antibiotics has necessitated reintroducing colistin as the last treatment option. However, the uncontrolled consumption of colistin, particularly for multidrug-resistant Gram-negative infections, has contributed to a surge in colistin resistance in many countries including Egypt. The present study aimed to determine colistin resistance among Gram-negative bacilli isolated from diverse clinical specimens. Methods: A total of 250 Gram-negative bacilli were included in the study. Antibiotic sensitivity for all isolates were performed using the Kirby-Bauer disc diffusion method. Colistin resistance was assessed by determination of minimal inhibitory concentration by broth microdilution method. Results: Out of 250 isolates, 36% were multidrug-resistant (MDR), with Escherichia coli being the most predominant MDR isolates (68.4%), while 55.2% were extensively drug-resistant (XDR) with the predominance of Acinetobacter baumannii (71%). Resistance to colistin was reported in 22.8% of all studied isolates. Colistin resistance among MDR isolates was 10% and 30.4% among XDR isolates. The highest colistin resistance was observed among Acinetobacter baumannii (73.7%) followed by Klebsiella pneumoniae (12.3%), then  Pseudomonas aeruginosa, and Escherichia coli (each 7%). The colistin-resistant isolates exhibited high resistance to ß-lactams antibiotics including 3^rd and 4^th generation cephalosporins (96.5%, 89.5% respectively), ciprofloxacin (80.7%), and aminoglycosides (71%). The isolates showed maximum sensitivity to doxycycline (58.5%), and sensitivity to imipenem and meropenem was 26%. Conclusion: High rates of MDR and XDR were observed among the recovered Gram-negative bacilli isolates. Colistin resistance was alarming in this study.