There is a dearth of empirical research on the attitudes of healthcare professionals toward trans and gender populations in Africa, particularly concerning the influence of socio-demographic and training-related factors. This gap limits the development of inclusive, affirming mental health services for trans and gender individuals in South Africa. The study that informs this article examined how healthcare professionals' socio-demographic characteristics and training experiences influence their attitudes toward trans and gender diverse clients in South Africa. It also explored which specific variables are associated with more positive or negative dispositions. A quantitative, survey design was used. 432 participants were engaged in the study including registered psychologists, psychiatrists, psychiatric nurses, social workers, and other mental health professionals. Data were collected via Qualtrics and analysed using a multi-level one-way analysis of variance in IBM SPSS Statistics (version 23). Findings revealed that among the provinces of South Africa, professionals residing in the Western Cape, KwaZulu-Natal and Gauteng; professionals in middle adulthood; professionals self-identifying as pansexual, lesbian or queer; clinical social workers, clinical psychologists, psychiatric nurses and psychiatrists; professionals with a master's, doctoral, of honours degree; professionals employed in the higher education/school sector, multi-sectors, or in private practice; professionals with a history of LGBT+-related research, those who completed a LGBT+-specific course, or had exposure to LGBT+ content during their clinical training; and respondents with more than 10 LGBT family members, friends or acquaintances exhibited the most positive attitudes towards trans and gender diverse clients when compared to counterparts in respective categories. The study concluded that provincial location, age, sexual orientation, occupation, education, training experience, and interpersonal relationships with LGBT+ populations play in shaping healthcare professionals' attitudes toward trans and gender diverse individuals in South Africa. Interestingly, some factors, like sex assigned at birth and years of experience, showed no significant influence.