Black adolescent girls aged between 14 and 19 years are more likely than White girls to be diagnosed with a sexually transmitted infection (STI). As STI diagnosis is associated with an increase in the risk for HIV acquisition, an early intervention specifically tailored to Black adolescent girls is warranted. A web-based video game intervention has the potential to reach this demographic. Because studies of social and behavioral determinants of disease demonstrate the protective role of peer group structures on individual outcomes, a multiplayer game can facilitate opportunities to exchange and evaluate information, learn social norms, develop behavioral skills, and allow peers to influence attitudes and behavior. No prior research has examined the feasibility of a web-based multiplayer game intervention for this population. This study describes the protocol for a randomized controlled trial (RCT) pilot-testing the feasibility, acceptability, and limited efficacy of a multiplayer game-based intervention for increasing HIV and STI testing and condom use in Black adolescent girls. We enrolled 79 Black adolescent girls aged 14 to 19 years residing in the United States into a 2-arm parallel RCT. The intervention is a theory-based, community-informed, multiplayer game that can be played with peers on the web using videoconferencing software. The goal of the game is to empower Black adolescent girls to make healthy decisions regarding dating, relationships, and sex, thus reducing HIV and STI infection. Control condition participants received a list of resources after playing a time and attention control game. All study procedures were conducted via the internet. We conveniently sampled Black adolescent girls using web-based advertisements. Study assessments occurred at enrollment, 1 week, 1 month, and 4 months after enrollment. The primary outcome of this study is increased HIV and STI testing by Black adolescent girls. Secondary outcomes include increased condom use, self-efficacy to use condoms, positive attitudes toward condom use, intentions, harm perceptions, HIV and STI and pre-exposure prophylaxis knowledge, positive sexual norms, sexual communication with partners, and reduced incidence of sexual risk behaviors associated with HIV and STI transmission. Secondary outcomes also included assessment of intervention feasibility and acceptability. From February to April 2022, a total of 79 Black adolescent girls were enrolled, with 40 (51%) having been randomized into the intervention condition and 39 (49%) into the control condition. At baseline, participant ages ranged from 14 to 19 (mean 16.4, SD 1.23) years. Web-accessible game interventions overcome common impediments of face-to-face interventions presenting a unique opportunity to reach Black adolescent girls and improve their sexual health and self-efficacy. Trial data will provide information about the limited efficacy of the intervention and inform future web-based studies and a larger RCT aimed at improving the sexual health of Black adolescent girls. ClinicalTrials.gov NCT04108988; https://clinicaltrials.gov/ct2/show/NCT04108988. DERR1-10.2196/43666.