Background Sensitisation training can reduce judgemental and discriminatory attitudes amongst healthcare workers. The ‘Integrated Key Populations Sensitivity Training Programme for Healthcare Workers in South Africa’ aimed to improve access to appropriate and non-judgemental health services for ‘key populations’, specifically men who have sex with men, sex workers and people who use drugs, through the sensitisation of healthcare workers. Objectives The aim of this study was to evaluate the effects of the integrated key population sensitisation training intervention for healthcare workers, conducted between 2013 and 2014 in South Africa. Methods This study used a combination of qualitative and quantitative methods. Qualitative methods compared attitudes between healthcare workers who received the training intervention and those who did not. Quantitative methods were used to compare similar changes in awareness amongst healthcare workers before and after receiving the training. We explored shifts in attitudes towards key populations, changes in awareness of health issues related to stigma, discrimination, and changes in capacity to manage sexual health and HIV risk behaviours, including substance use and anal sex. Results The findings indicate that the training intervention resulted in a shift in attitudes, increased empathy for key populations, a reduction in negative and discriminatory moral-based judgements towards key populations and their behaviours, and increased self-perceived capacity to provide appropriate health services to key populations. Over 70% of healthcare workers trained in this programme strongly agreed that this intervention helped to increase awareness of psychosocial vulnerabilities of key populations, and address stigmatising attitudes. Conclusion The findings suggest that sensitisation training increases healthcare workers’ knowledge and awareness about specific HIV-related health needs and psychosocial vulnerabilities of key populations, reduces moralising and judgemental attitudes, and results in healthcare workers feeling more skilled to provide appropriate and sensitive services.