Background: Human Immunodeficiency Virus (HIV) is one of the world's top causes of death. New HIV infections emerge every day, leading HIV patients to seek care at health facilities and prompting health care professionals to undertake risky invasive procedures. And notwithstanding the mediation of science, this poses a risk of occupational exposure among health care workers (HCWs), hence the need for the effective use of HIV post-exposure prophylaxis (PEP). The aim of this study was to assess the knowledge, attitude and utilization of PEP among health care workers at HIV treatment centers in Port-Harcourt metropolis. Methodology: This descriptive cross-sectional study utilized a pre-tested, structured, self-administered questionnaire on a sample of 204 HCWs chosen by multi-stage sampling method. Data were analyzed and presented using descriptive and analytical statistics. Results: The study revealed that 39.7% of the respondents had good knowledge of HIV PEP and 96.5% had good attitude towards HIV PEP. Additionally, 22.1% had previously encountered possible occupational exposure to HIV, and only 45.5% of them took PEP. This translates to an overall PEP use of 10.1%. Significant associations were observed between knowledge and attitude towards PEP (p=<0.001), source of information and knowledge (p=<0.001), and source of information and attitude (p=0.02). The study also showed that sex, marital status and designation was associated with utilization of PEP (p=0.01; p=0.04; p=0.02). Conclusion: The study revealed low utilization of PEP despite the level of good and fair knowledge and a generally positive attitude towards PEP. There exists a gap between knowledge and utilization of PEP, hence the need for periodical retraining of HCWs. This should be supplemented by ensuring the consistent availability and accessibility of PEP at treatment centers.