Men who have sex with men (MSM) account for more than two thirds of new HIV diagnoses annually. Sexual behavior that increases risk for onward transmission of HIV is associated with both alcohol and cannabis use. However, little is known about the influence of cannabis and alcohol co-use on engagement in condomless anal sex (CAS). The current study explored daily associations between substance use and CAS among HIV-positive MSM using a 42-day timeline followback interview (N = 101). Generalized estimating equation (GEE) logistic regression models were used to examine the association between cannabis and alcohol co-use and CAS at the sexual event while controlling for study site, condition, adherence to antiretroviral therapy, sex-related alcohol expectancies, and partner type. Participants provided data for 1052 sexual activity days, 60.7% of which involved CAS. Of 638 CAS days, 9.1% involved no substances, 72.0% involved either cannabis or alcohol use, and 18.9% involved cannabis and alcohol co-use. Results indicated that the odds of engaging in CAS were higher for sexual events in which cannabis and alcohol co-use occurred (aOR 2.98; 95% CI 1.27, 6.97) compared to events in which no substance use occurred (p = 0.012), but this relationship was no longer significant when cannabis and alcohol co-use was compared to single substance use (aOR 1.57; 95% CI 0.85, 2.90; p = 0.15). Future research should identify specific substance use (e.g., quantity) and partner characteristics (e.g., level of intoxication) that may uniquely influence the relationship between cannabis and alcohol co-use and condomless sex.