Aim: This study aimed to investigate the long-term monitoring of hospital-acquired infections (HAIs), focusing on airborne and surface microbial contaminants in a tertiary care hospital. The objective was to assess the microbial load in various hospital environments and explore its correlation with the incidence of HAIs. Materials and Methods: A cohort of 120 patients, including ICU (n=30), general wards (n=60), and emergency department (ED) (n=30), was enrolled. Microbiological samples from patients (swabs, urine, blood, sputum) and environmental samples (surfaces and air) were collected. The samples were cultured on appropriate agar plates, and antimicrobial resistance testing was performed using the disk diffusion method. Data were analyzed using descriptive statistics and correlation analyses. Results: The study revealed that the ICU had the highest microbial contamination across both surfaces and airborne samples. The prevalence of pathogens such as Staphylococcus aureus (20.8%) and Escherichia coli (12.5%) was significant in both patient and environmental samples. The ICU showed the highest incidence of HAIs (56.7%) and the longest length of stay. The antimicrobial resistance rates for Staphylococcus aureus (29.17%) and Escherichia coli (23.33%) were notably high, highlighting a concerning trend of multidrug resistance. Conclusion: This study underscores the elevated risk of HAIs in ICU settings, which are associated with higher microbial contamination and antimicrobial resistance. The findings emphasize the need for rigorous infection control measures, particularly in critical areas like the ICU, to reduce the burden of HAIs. Enhanced environmental cleaning and targeted antimicrobial stewardship are critical to improving patient outcomes in hospital settings.