Introduction: Nosocomial infections (NI) are common conditions among hospitalized patients. This study aimed to evaluate healthcare workers' knowledge and practices regarding infection prevention measures in intensive care units. Method: This was a descriptive, cross-sectional study conducted from April 13to June 10, 2022, among 45 healthcare workers in three university teaching hospitals of Abidjan. The parameters studied were: the theoretical and practical knowledge of healthcare workers regarding the prevention of nosocomial infections. Results: 45 healthcare workers participated. 74.7% knew the definition of a nosocomial infection and 43% identified the main risk factors for its occurrence. Sixty-five percent identified staff hands as the main mode of cross-transmission of germs between patients. Sixty-four percent of staff reported using non-sterile gloves during urinary catheter insertion. Compliance with hand hygiene before patient contact was low (31.8%) while gloves changes between patients were observed in 87.2% of cases. Deficiencies were noted in adherence to aseptic techniques before performing invasive procedures. Non-compliance was mainly attributed to shortages of supplies and personnel, and insufficient knowledge of preventive measures. Conclusion: healthcare workers’ knowledge and adherence to hygiene practices in intensive care units were insufficient.