Reducing the stigma and discrimination faced by men who have sex with men (MSM) and transgender women (TGW) in healthcare settings is key to improving health outcomes. Using a one-group pre- and post-test design, we tested the efficacy of a theory-informed, multi-level pilot intervention (“Harmony”) among 98 healthcare workers (HCWs) to reduce sexual orientation and gender identity (SOGI)-related stigma and discrimination faced by MSM and TGW in two public hospitals. The intervention contained group-level (a half-day workshop) and individual-level (four videos) components. Using multi-level modelling, we compared knowledge, attitudes, and comfort level among HCWs across three timepoints: pre-intervention, post-intervention, and follow-up (2 months after the intervention). Client surveys were conducted among 400 MSM/TGW (two independent samples of 200 MSM/TGW) attending the intervention hospitals, before the intervention among HCWs and three months after the intervention. Generalised estimating equations assessed service users’ satisfaction with hospital services, discrimination experiences, and positive interactions with HCWs. Significant changes were observed in primary outcomes: 30% increase in positive attitude scores (incidence rate ratio (IRR) = 1.30, 95% CI 1.13–1.49) and 23% increase in the proportion of HCWs reporting being comfortable in providing care to MSM/TGW (IRR = 1.23, 95% CI 0.03–1.68). Similarly, there was a significant improvement in secondary outcomes (scores): support for non-discriminatory hospital policies (IRR = 1.08, 95% CI 1.004–1.15), the importance of asking SOGI questions in clinical history (IRR = 1.17, 95% CI 1.06–1.29), and perceived self-efficacy in providing clinical care (IRR = 1.13, 95% CI 1.01–1.27). Service users’ data provided corroborative evidence for intervention efficacy: e.g., 14% increase in the proportion of MSM reporting overall satisfaction with hospital services and 6% and 15% increase in the scores of positive interactions with HCWs in the combined sample of MSM/TGW and TGW, respectively. The Harmony intervention showed preliminary evidence for improving positive attitudes, comfort level, and understanding of the healthcare issues of MSM/TGW among HCWs, warranting large-scale implementation research.