Aim: The study aimed to determine the prevalence of Methicillin-Resistant Staphylococcus aureus (MRSA) colonization among healthcare workers (HCWs), identify associated risk factors, and analyze the antibiotic susceptibility patterns of MRSA isolates in a tertiary care hospital setting. Materials and Methods: This cross-sectional observational study included 120 HCWs from various professional categories, including doctors, nurses, laboratory staff, ward attendants, and housekeeping staff. Nasal swabs were collected from all participants under aseptic conditions and processed on Mannitol Salt Agar (MSA). Identification of Staphylococcus aureus was confirmed using Gram staining and biochemical tests. Methicillin resistance was detected using the Cefoxitin Disk Diffusion Test (30 µg) on Mueller-Hinton Agar (MHA), following CLSI guidelines. Data on demographic details, risk factors, and clinical history were collected through a structured questionnaire and analyzed using SPSS Version 16.0, with a significance level of p < 0.05. Results: Among the 120 participants, nurses constituted the largest group (33.33%), followed by doctors (25.00%) and other staff categories. Staphylococcus aureus was isolated in 41.67% of samples, with 16.67% identified as MRSA. Nurses had the highest MRSA carriage rate (20.00%). Key risk factors included poor hand hygiene (50.00%), history of skin infections (25.00%), and recent antibiotic use (15.00%). Antibiotic susceptibility testing showed 100% sensitivity to Vancomycin, while Erythromycin resistance was observed in 50% of isolates. Conclusion: The study revealed a significant burden of MRSA colonization among healthcare workers, with nurses being the most affected. Poor hand hygiene practices and antibiotic misuse emerged as major risk factors. Vancomycin remains highly effective against MRSA isolates, emphasizing the need for regular MRSA screening, robust infection control measures, and effective antibiotic stewardship programs to mitigate MRSA transmission in healthcare settings.