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Background: Dengue fever is a significant public health concern. The advent of social media has introduced digital opinion leaders (DOLs), health care professionals with substantial online followings who play a pivotal role in disseminating health information and combating misinformation. Objective: We aimed to investigate the role of DOLs in dengue prevention and explore their preferences for collaboration with health sector entities to strengthen dengue prevention initiatives. Methods: A qualitative study was conducted using semistructured interviews with 37 purposively selected DOLs from 8 countries in Latin America and Southeast Asia. They were selected based on their active online presence, dissemination of dengue-related content, and substantial social media followings. Interviews took place either in person or online, according to the participants’ chosen languages. Each session, lasting approximately 60 minutes, was audio recorded, transcribed verbatim, and subjected to thematic analysis to identify recurring themes. Results: The thematic analysis led to several key findings. First, DOLs used social media to enhance public health communication, focusing on raising awareness (16/37, 43%), correcting misconceptions (17/37, 46%), and modeling preventive behaviors (8/37, 22%) for infectious diseases. They educated audiences on disease symptoms and prevention, addressed vaccine hesitancy, and shared personal practices to encourage similar actions among followers. Second, 35% (13/37) of the DOLs reported a widespread lack of public knowledge about dengue and its prevention, with even less awareness of vaccine availability. In addition, 27% (10/37) of them identified challenges due to antivaccination sentiments and misinformation, while 8% (3/37) noted obstacles from perceived inadequate government leadership in dengue prevention. In response, DOLs leveraged their social media influence to educate the public. A significant number (22/37, 59%) of the DOLs emphasized the importance of regular promotion of vector control measures as the cornerstone of dengue prevention and 68% (25/37) highlighted the critical role of vaccines, particularly among vulnerable groups. Finally, collaboration was essential for expanding DOLs’ reach and credibility, with 54% (20/37) of them partnering with pharmaceutical companies, 43% (16/37) with government agencies, and 27% (10/37) with nongovernmental organizations. In these collaborations, 38% (14/37) of the DOLs emphasized the importance of adhering to ethical standards, and 57% (21/37) prioritized projects aligning with their personal values and professional standards, avoiding producing content that contradicted their beliefs or goals. Conclusions: DOLs are essential in disseminating dengue prevention information. They recognize their responsibility to raise awareness about dengue vaccines and dispel related misconceptions to combat vaccine hesitancy. Unlike nonmedical social media influencers, whose content may lack medical accuracy and be driven by monetization, DOLs provide evidence-based information, enhancing their credibility. Collaborations between DOLs and health sector stakeholders, although currently limited, hold significant potential for effective dengue prevention, provided they adhere to ethical standards and are supported by credible scientific evidence.