Background: Hospital-acquired infections (HAIs) pose a significant challenge to healthcare systems globally, exacerbated by rising antimicrobial resistance. Understanding the prevalence, distribution, and resistance patterns of pathogens causing HAIs is critical for guiding effective infection control and antibiotic stewardship strategies. Methods: This cross-sectional study was conducted in a tertiary care hospital, involving 1,780 clinical samples collected from patients who developed infections 48 hours or more after admission. Samples were obtained from multiple hospital wards including surgery, internal medicine, intensive care units, and transplant units. Microbial identification and antimicrobial susceptibility testing were performed according to CLSI guidelines. HAIs were categorized by infection type and analyzed for resistance profiles. Statistical associations between resistance patterns and clinical variables such as age, gender, ward type, infection detection time, length of hospital stay, and patient outcomes were evaluated. Results: Among the isolates, Gram-negative bacteria predominated (58.1%), chiefly Escherichia coli and Klebsiella pneumoniae, followed by Gram-positive organisms (21.2%) such as Enterococcus spp. and Staphylococcus aureus. Urinary tract infections were the most common HAI. High rates of antimicrobial resistance were observed, including methicillin-resistant S. aureus (52.7%), vancomycin-resistant Enterococcus spp. (58.4%), extended-spectrum beta-lactamase-producing K. pneumoniae (74.8%), and notable carbapenem resistance among Acinetobacter spp. (93.4%). Significant associations were identified between resistant infections and variables such as ICU admission, gender, and infection detection timing. Conclusion: This study highlights the substantial burden of multidrug-resistant pathogens in hospital-acquired infections, particularly in high-risk hospital wards. Enhanced infection control measures and targeted antimicrobial stewardship are essential to mitigate the spread of resistance and improve patient outcomes.