Purpose International studies document that lesbian, gay, bisexual, transgender, queer and intersex (LGBTQI+) patients face significant health disparities. Studies exploring the attitudes, knowledge, preparedness and comfort levels of healthcare students towards LGBTQI+ health have been conducted in the United States, United Kingdom and Malaysia. This study aims to investigate stigma in healthcare for LGBTQI+ patients in Singapore, and possible upstream factors within medical education. Methods This mixed-methods study adopts a convergent parallel design. The Health Stigma and Discrimination Framework was referenced to devise in-depth interviews with representatives from 13 LGBTQI-affirming non-governmental organisations, analysed through thematic analysis. 320 clinical medical students were surveyed about attitudes, knowledge, comfort, preparedness, and perceived importance of/towards LGBTQI+ health, analysed via descriptive statistics and multivariate regression. Results Prevailing stigma in Singaporean society against LGBTQI+ individuals is exacerbated in healthcare settings. Doctors were cited as unfamiliar or uncomfortable with LGBTQI+ health, possibly from lack of training. Among medical students surveyed, the median composite attitudes, comfort and preparedness index was 3.30 (Interquartile Range (IQR) = 0.50), 3.17 (IQR = 0.83), 2.50 (IQR = 1.00) respectively. Only 12.19% of students answered all 11 true-false questions about LGBTQI+ health correctly. Conclusion Medical students in Singapore have scored sub-optimally in their knowledge and preparedness towards LGBTQI+ health, while interpersonal and structural stigma in healthcare towards LGBTQI+ people in Singapore negatively affects health and wellbeing. These findings are an impetus to improve medical training in this area. High scores among medical students in attitudes, comfort and perceived importance of LGBTQI+ topics demonstrate that there is space for LGBTQI+ health in the local medical education curriculum. Curricular interventions can prioritise content knowledge, communication skills and sensitivity.