Background Gay, bisexual, and other men who have sex with men and transgender individuals are more heavily affected by HIV and other sexually transmitted infections (STIs) than their cisgender, heterosexual peers. In addition, sexual and gender minorities who use substances are often at a further increased risk of HIV and other STIs. Increasing testing for HIV and other STIs allows this hardly reached population to receive early intervention, prevention, and education. Objective We explored HIV and STI testing patterns among 414 sexual and gender minority adolescents and young adults aged 15 to 29 years who self-reported substance use and lived in southeastern Michigan. Methods We analyzed data from the baseline survey of a 4-arm randomized controlled trial that aimed to examine the efficacy of a brief substance use intervention for creating gains in engagement in HIV prevention. We fit multinomial logistic regression models to 2 categorical HIV and STI testing variables (lifetime and previous 12 months) based on self-reports of testing (never, STIs only, HIV only, or both). In addition, we compared HIV and STI testing behaviors across demographic characteristics, structural factors, psychosocial barriers, substance use, and sexual behaviors. Results Our findings showed that 35.5% (147/414) of adolescents and young adults reported not being tested for either HIV or STIs in the previous year, and less than half (168/414, 40.6%) of the sample achieved the Centers for Disease Control and Prevention recommendation of HIV and STI testing once per year. We observed HIV and STI testing disparities across sociodemographic (eg, sexual identity, education, and income) and health (eg, substance use) correlates. Specifically, cisgender gay men who have sex with men were more likely to report being tested for HIV compared with bisexual men and transgender individuals, who were more likely to be tested for STIs. Conclusions This study illustrates the results of an HIV prevention intervention in southeastern Michigan showing the need for HIV prevention interventions that leverage structural factors, psychosocial barriers, and substance use as key drivers to achieve HIV and STI testing rates to meet the Centers for Disease Control and Prevention guidelines. Trial Registration ClinicalTrials.gov NCT02945436; http://clinicaltrials.gov/ct2/show/NCT02945436 International Registered Report Identifier (IRRID) RR2-10.2196/resprot.9414