Protecting the rights of the lesbians, gays, bisexuals, transgender, intersex, and queers (LGBTIQ) population requires, first and foremost, a proper understanding of their sexual orientation and gender identity. This study highlights a severe misunderstanding and lack of knowledge among health professionals in Vietnam with regard to the men who have sex with men (MSM) and transgenders. This study uses (i) a survey based on the convenience sampling method among 150 health workers that covered 61 questions and (ii) 12 in-depth interviews in two metropolitan centres in Vietnam, Hanoi and Ho Chi Minh city. Three main topics are explored: (i) the general knowledge of healthcare workers about MSM and transgenders; (ii) their knowledge about the sexual reproductive health and human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) risks of MSM and transgenders; and (iii) their attitudes and behaviors towards MSM and transgenders. One of the notable findings is how prevalent the misperceptions are across the board, namely, in staff of both sexes, in both cities, at various kinds of medical facilities, at different work positions and educational levels. Half of the respondents consider transgenders to have a curable mental problem while 45% say MSM only have sex with males. Most remarkably, 12.7% state if they have any choice, they want nothing to do with MSM and transgenders. The study finds there is a considerable percentage of health professionals who lack knowledge about the diversity of sexual orientation, gender identity, and health issues related to the sexual minorities and gender non-conforming population. To improve the clinical process for serving these at-risk groups, the study suggests the continual education for the health workers needs to be added to their formal as well as in-job training.