Background: Stigma and discrimination are among the greatest challenges that face people living with human immunodeficiency virus (PLHIV), affecting their health provision. This might harm the overall retention of patients and the effective care and control of the disease. Hence, our study aimed to explore the knowledge, attitude, and practice (KAP) of HCWs in tertiary hospitals in Zagazig City, as regards HIV prevention and healthcare provision to PLHIV. Methods: A cross-sectional study was conducted in a convenience sample of 295 doctors and nurses from Tropical and Internal Medicine Departments in two tertiary care hospitals in Zagazig City, using a self-administered questionnaire designed to explore the KAP of HCWs, as regards HIV prevention and healthcare provision to HIV- infected people. Results: This study revealed that the HCWs had good knowledge about the nature and mode of transmission of HIV. Still, they needed a more detailed understanding of the disease to prevent stigmatization and discrimination of health provision. Moreover, there were significant distinctions between physicians and nurses in their participation in training programs and their performance across the KAP domains. Conclusion: This study can provide a benchmark for further studies in Egypt aiming to set up a training program to enhance HCWs' knowledge regarding HIV and eradicate stigmatizing attitudes and behaviors.