Colistin, often referred to as a "salvation drug," is critical for treating severe infections caused by multidrug-resistant Gram-negative bacteria (MDRGNB), is experiencing an alarming increase in resistance worldwide. This study aims to elucidate the prevalence of colistin resistance across various drug-resistant phenotypes, specifically Usual drug Resistant (UDR) and Difficult-to-Treat (DTR), as well as among different antibiotic-class resistant categories, including Carbapenem-resistant (CR), Fluoroquinolone-resistant (FQR), and Extended-spectrum Cephalosporin-resistant (ECR) pathogens. Conducted across multiple healthcare settings in Egypt, we employed Vitek 2 for isolate identification and selective media for screening colistin resistance. The minimum inhibitory concentration (MIC) of colistin was determined using broth microdilution (BMD), and PCR assays were conducted to detect plasmid-mediated mcr-1 and mcr-2 genes. Among 150 isolates, a concerning 78% demonstrated resistance to colistin, with significant occurrences in urinary tract infections (33%), respiratory tract infections (32%), and bloodstream infections (19%). The predominant colistin-resistant organisms identified included Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. Importantly, none of the isolates harbored the mcr-1 gene, and only one E. coli isolate harbored the mcr-2 gene. This research underscores the urgent need for enhanced surveillance and targeted therapeutic strategies, revealing the escalating incidence of colistin resistance among diverse drug-resistant Gram-negative pathogens, which presents a formidable challenge to effective clinical management.