BACKGROUND: HIV testing coverage among males having sex with males (MSM) and transgender women (locally known as ) is low in Bangladesh. Oral fluid-based HIV self-testing (HIVST) may improve coverage due to its convenience and privacy but is yet to be tested in Bangladesh. Therefore, the acceptability and feasibility of supervised HIVST was examined. METHODS: A cross-sectional study was conducted among 379 MSM and selected from five geographical regions from February-October 2020. Semi-structured questionnaire was used to examine socio-demographics, risk behaviors, and perception to acceptability and feasibility (correct completion) of HIVST. Both bivariate and multivariable logistic regression analyses were performed. Qualitative data collection encompassed in-depth interviews (N = 19), key informant interviews (N = 10), and two focus group discussions (N = 12). Line-by-line content, contextual and thematic analysis were done and triangulated to explore facilitators and challenges of HIVST among MSM and . RESULTS: Among 379 participants, the acceptability of HIVST was 99.5 % (n = 377). Reasons for acceptability included interest in independent testing (84.3 %), peer influence (57.3 %), quicker-easier procedure (54.9 %), and painless procedure (52.5 %). Qualitative findings revealed participant's risk perceptions, empowering feelings, social stigma, complementing working hours, and convenience during COVID-19 lockdowns. Around 92 % of the participants correctly completed HIVST. In multivariable analysis, the likelihood of correct test conduction was found higher among metropolitan, younger, married, educated, and participants who felt confident during HIVST process. Qualitative findings underscored the importance of supervising the use of HIVST for first-time users. Participants, particularly the less educated groups, highlighted the video demonstration as a useful tool in the context of difficulties in reading the textual instructions. However, most participants pointed out the result interpretation as the trickiest part of HIVST. All participants demonstrated willingness for future HIVST, were interested in social media-based approaches (84 %), were willing to purchase subsidized kits, and preferred conducting future tests alone at home (83.2 %). CONCLUSION: Oral fluid-based HIVST was an acceptable and feasible approach for MSM and in Bangladesh who were willing to do future tests independently after the supervised approach. Willingness to purchase kits and interest for social media-based approaches indicate scalability and sustainability potential of HIVST.