Central venous catheters are commonly inserted to monitor patients with critical illnesses. Even when used to treat very ill patients, they are susceptible to widespread headaches, including central line-associated bloodstream infections (CLABSI). Central line-associated bloodstream infection (CLABSI) is one of the most significant HAIs, associated with excess mortality of 12–25%. To discover CLABSI cases, microbiological profiles, and their antimicrobial susceptibility. The study was conducted in an intensive care unit over a period of 12 months. 150 blood samples and catheter tips were collected for the culture of suspected or secondary bacteremia. CLABSI is described as being consistent with the CDC’s proposal. Automated VITEK 2 technology identifies bacterial isolates and investigates their antimicrobial susceptibility. Out of 150 samples, 50 showed no growth, 45 showed colonizers, 40 showed CLABSI, and 15 showed secondary infection. Fifty-five had positive blood cultures, 15 of whom had another source of infection. In our study, the CLABSI rate was 7.8/1000 central venous days. Rigorous implementation of the system and maintenance of the central line bundle are mandatory to prevent colonization.