BACKGROUND: There are very few studies in Saudi Arabia on stigmatization and discrimination against people living with HIV/AIDS (PLWHA), a critical step for the prevention and control of HIV. The aim of this study was to assess the level of stigmatization and discrimination against PLWHA by healthcare workers (HCWs) as well as their knowledge, attitude, and practices (KAP). MATERIALS AND METHODS: This cross-sectional study included 182 HCWs at primary healthcare centers in Medina, Saudi Arabia. The validated short version of the Healthcare Provider HIV/AIDS Stigma Scale and the AIDS Attitude Scale were used in this research. Student’s t-test and analysis of variance (ANOVA) were used to assess the differences in the mean knowledge, attitudes, and practices KAP scores by various sociodemographic factors. Multiple linear regression analysis was used to determine factors associated with KAP scores. RESULTS: Most participants were males (58.2%) and aged more than 30 years (60.4%). The tendency to stigmatizing behavior was present in 24.2%–68.17% of the participants, and discriminatory practice was present in 11.5%–50% of the participants. In multiple linear regression analysis, factors that independently predicted the knowledge score were being a doctor compared to nurses (P < 0.001), receiving in-service training for PLWHA (P < 0.001), and male gender (P = 0.002). Attitude was predicted by being female (P = 0.008) and a doctor (P = 0.005). Practice was predicted by the knowledge score (P < 0.001) and being married (P = 0.035). CONCLUSION: This study found that stigmatization and discrimination were less prevalent in HCWs who had good HIV-related knowledge and had received in-service training for PLWHA. The results highlight the significance of continuing education and training opportunities for HCWs to provide effective and appropriate treatment to PLWHA.