The stigmatizing attitude towards people living with HIV/AIDS (PLWHA) can be a major barrier to effective patient care. As future physicians, medical students represent a core group that should be targeted with focused knowledge and adequate training to provide patient care without prejudice. The aim of the current study was to examine HIV/AIDS knowledge, and the stigmatizing attitude towards PLWHA, among medical students in Jordan. The current study was based on a self-administered online questionnaire, which was distributed during March–May 2021, involving students at the six medical schools in Jordan, with items assessing demographics, HIV/AIDS knowledge, and HIV/AIDS stigmatizing attitude, which was evaluated using the validated HIV-stigma scale. The total number of respondents was 1362, with predominance of females (n = 780, 57.3%). Lack of HIV/AIDS knowledge among the study participants was notable for the following items: HIV transmission through breastfeeding (40.8% correct responses), HIV is not transmitted through saliva (42.6% correct responses), and vertical transmission of HIV can be prevented (48.8% correct responses). Approximately two-thirds of the respondents displayed a positive attitude towards PLWHA. For six out of the 14 HIV/AIDS knowledge items, lack of knowledge was significantly correlated with a more negative attitude towards PLWHA. Multinomial regression analysis showed that a significantly more negative attitude towards PLWHA was found among the pre-clinical students compared to the clinical students (odds ratio (OR): 0.65, 95% confidence interval (CI): 0.43–0.97, p = 0.036); and that affiliation to medical schools that were founded before 2000 was associated with a more positive attitude towards PLWHA compared to affiliation to recently founded medical schools in the country (OR: 1.85, 95% CI: 1.42–2.42, p < 0.001). About one-third of medical students who participated in the study displayed a negative attitude towards PLWHA. Defects in HIV/AIDS knowledge were detected for aspects involving HIV transmission and prevention, and such defects were correlated with a more negative attitude towards PLWHA. It is recommended to revise the current medical training curricula, and to tailor improvements in the overall HIV/AIDS knowledge, which can be reflected in a more positive attitude towards PLWHA, particularly for the recently established medical schools in the country.