The healthcare needs of lesbian, gay, bisexual, trans*, queer, and intersex (LGBTQI+) persons are often overlooked, prompting national and international calls to include diversity-related competencies into medical students’ training. However, LGBTQI+-focused healthcare education targets remain elusive, as surveys reveal considerable variability across national student populations. To generate empirical data and vocalize recommendations for medical education, we conducted the first nationwide online survey among 670 German medical students from 33 universities. Overall, most respondents reported low confidence regarding their medical training preparing them for LGBTQI+ patients, stated that LGBTQI+ themes were not covered during training, and agreed that the inclusion of such themes is urgently needed. In addition, we found gender and LGBTQI+ community member status to be key variables. Men scored lower in knowledge than women, while community members scored higher than non-community members. Similarly, community members reported higher comfort levels. Non-community men showed the highest levels of prejudice and efficacy beliefs, while at the same time had the lowest scores in contacts and the perceived importance of LGBTQI+-related teaching. Keeping subgroup differences in mind, we recommend that educational training should include LGBTQI+ healthcare aspects and address self-efficacy beliefs in future medical professionals to overcome LGBTQI+ healthcare disparities.