Objective: Evaluate the utility of brief online training for facilitating mental health professionals’ perceived knowledge, confidence, and comfort in working with non-binary clients, and building positive attitudes. Method: Baseline participants were 79 Australian registered mental health professionals, of whom 38 completed online training and a one-week follow-up assessment. Participants completed an assessment of attitudes, comfort, confidence, and perceived knowledge pertaining to non-binary people before and after completing online training. The training (30-minute average completion time) provided information on non-binary people’s lives using text, images, embedded video, and links to optional resources. Results: Follow-up measures completed one-week post-intervention indicated engagement in the training may lead to increased perceived knowledge, confidence and comfort. Attitudes were positive at baseline and were not significantly different at follow-up. Conclusions: Providing brief online training for working with non-binary people in mental health might improve practitioner perceived knowledge, confidence and competence at low cost and with easy access. Future research is required to account for limitations and build on current outcomes. Key Points What is already known on this topic: Little is known about the effects of brief online training regarding non-binary genders on mental health practitioner’s attitudes, confidence, comfort, and perceived knowledge. Previous research has shown that brief training can increase positive attitudes for, knowledge of, behavioural intent, and cultural competence for working with sexual and gender minorities, though has not specifically focused on non-binary people. Previous literature has highlighted positive associations between practitioner knowledge and the well-being of non-binary people accessing health services. What this paper adds: This paper adds to the ongoing genesis of options for training mental health practitioners about non-binary people and relevant issues. Findings suggest beneficial outcomes from both prior professional training in this area and the brief training used in the study. The brief online training used in the study was associated with higher perceived knowledge, confidence, and comfort for working with non-binary clients, but further research is needed to understand reasons for change in these variables.