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<b>Background/Objectives:</b> Stigma and fear related to human immunodeficiency virus persist in healthcare settings and negatively influence professionals' attitudes and the quality of care provided to people living with human immunodeficiency virus. This study aimed to evaluate knowledge, attitudes, and stigma toward people living with human immunodeficiency virus among healthcare professionals in Spain and to explore strategies to reduce stigma. <b>Methods:</b> An explanatory sequential mixed-methods study was conducted. In the quantitative phase, an online questionnaire based on the International Planned Parenthood Federation instrument was disseminated nationwide through social media using non-probability convenience sampling. Quantitative data from 1013 healthcare professionals were analyzed using descriptive statistics and non-parametric tests (Kruskal-Wallis, chi-square, Friedman) with appropriate corrections for multiple comparisons. In the qualitative phase, 12 participants were purposively selected for semi-structured interviews to explain quantitative findings. Qualitative data were analyzed using reflexive thematic analysis. Integration occurred through joint interpretation and a joint display table connecting quantitative patterns with qualitative themes. Ethical approval was obtained from the Clinical Research Ethics Committee of Bellvitge Hospital in Catalonia. <b>Results</b>: A total of 1013 healthcare professionals from diverse specialties participated, and twelve completed qualitative interviews. Knowledge regarding transmission, prevention, and treatment of human immunodeficiency virus was high. However, more than half reported no specific training and felt unprepared to care for people living with human immunodeficiency virus. Despite knowledge, fear of contagion was common. Attitudes were positive, with acceptance of caring for people living with human immunodeficiency virus and rejection of common misconceptions. Qualitative findings revealed persistent stigma linked to insufficient training and cultural prejudice. Integration of quantitative and qualitative data revealed that knowledge alone does not eliminate fear, and that the gap between theoretical understanding and clinical confidence represents a critical barrier to stigma-free care. <b>Conclusions:</b> Although healthcare professionals in Spain demonstrate knowledge about human immunodeficiency virus, stigma and fear remain prevalent. Targeted education and interprofessional training are needed to ensure respectful, inclusive, and stigma-free clinical care.