Suicide risk is a global public health crisis, with suicide ranking as a consistent leading cause of death among adults in the US. Autistic individuals and transgender or gender-nonconforming (TGNC) individuals represent populations with notably elevated rates of suicidal thoughts and behaviors (STBs). To characterize suicidal thoughts and behaviors among TGNC and autistic individuals, using a large, nationally representative sample. This study is a secondary analysis of cross-sectional data from students at colleges and universities throughout the US who participated in the American College Health Association National College Health Assessment from 2019 to 2023. Autistic and TGNC identities were self-reported by participants. The frequency of intersectionality of autism and TGNC identities and whether those who had intersectional marginalized identities had increased likelihood of STBs were examined. STBs were self-reported by participants. A series of moderated regression analyses were performed to examine how the interaction between autism and possessing a marginalized gender identity (ie, TGNC status) was associated with STBs. The sample included 41 507 college students with a mean (SD) age of 23.35 (6.83) years. A total of 2410 participants (5.81%) identified as being TGNC. Overall, 326 TGNC participants (13.53%) also identified as autistic, whereas 625 of those who identified as cisgender (1.58%) also identified as autistic. Gender identity and autism were associated with greater odds of STBs. For suicidal ideation, gender identity had an odds ratio (OR) of 3.34 (95% CI, 2.99-3.73), and autism had an OR of 2.06 (95% CI, 1.76-2.42). For suicide attempts, gender identity had an OR of 2.74 (95% CI, 2.13-3.52), and autism had an OR of 2.39 (95% CI, 1.62-3.52). A significant interaction existed for attempts (OR, 0.51; 95% CI, 0.27-0.97); nonautistic cisgender individuals had the lowest attempt rate. This cross-sectional study addresses the dearth of information on how intersectionality in gender and autism status impacts the risk of STBs, and the results confirm the elevated risk of STBs among TGNC and autistic populations. Interventions are needed to support college students with these identities.