BACKGROUND: Mobile technologies are being increasingly developed to support the practice of medicine, nursing, and public health, including HIV testing and prevention. Chatbots using artificial intelligence (AI) are novel mobile health strategies that can promote HIV testing and prevention among men who have sex with men (MSM) in Malaysia, a hard-to-reach population at elevated risk of HIV, yet little is known about the features that are important to this key population. OBJECTIVE: The aim of this study was to identify the barriers to and facilitators of Malaysian MSM's acceptance of an AI chatbot designed to assist in HIV testing and prevention in relation to its perceived benefits, limitations, and preferred features among potential users. METHODS: We conducted 5 structured web-based focus group interviews with 31 MSM in Malaysia between July 2021 and September 2021. The interviews were first recorded, transcribed, coded, and thematically analyzed using NVivo (version 9; QSR International). Subsequently, the unified theory of acceptance and use of technology was used to guide data analysis to map emerging themes related to the barriers to and facilitators of chatbot acceptance onto its 4 domains: performance expectancy, effort expectancy, facilitating conditions, and social influence. RESULTS: Multiple barriers and facilitators influencing MSM's acceptance of an AI chatbot were identified for each domain. Performance expectancy (ie, the perceived usefulness of the AI chatbot) was influenced by MSM's concerns about the AI chatbot's ability to deliver accurate information, its effectiveness in information dissemination and problem-solving, and its ability to provide emotional support and raise health awareness. Convenience, cost, and technical errors influenced the AI chatbot's effort expectancy (ie, the perceived ease of use). Efficient linkage to health care professionals and HIV self-testing was reported as a facilitating condition of MSM's receptiveness to using an AI chatbot to access HIV testing. Participants stated that social influence (ie, sociopolitical climate) factors influencing the acceptance of mobile technology that addressed HIV in Malaysia included privacy concerns, pervasive stigma against homosexuality, and the criminalization of same-sex sexual behaviors. Key design strategies that could enhance MSM's acceptance of an HIV prevention AI chatbot included an anonymous user setting; embedding the chatbot in MSM-friendly web-based platforms; and providing user-guiding questions and options related to HIV testing, prevention, and treatment. CONCLUSIONS: This study provides important insights into key features and potential implementation strategies central to designing an AI chatbot as a culturally sensitive digital health tool to prevent stigmatized health conditions in vulnerable and systematically marginalized populations. Such features not only are crucial to designing effective user-centered and culturally situated mobile health interventions for MSM in Malaysia but also illuminate the importance of incorporating social stigma considerations into health technology implementation strategies.