The mental health of gay and bisexual men (GBM) who use preexposure prophylaxis (PrEP) may be negatively impacted by experiences of stigma. To assess the relationship between stigma and mental health among GBM who use PrEP in Guatemala, we conducted a survey with 162 PrEP users recruited in a community-based PrEP clinic. In multiple linear regression analyses, greater internalized (β = 1.94, SE = 0.85, p = .023) and enacted sexual orientation stigma (β = 0.50, SE = 0.12, p < .001) were associated with greater depressive symptoms. Greater internalized (β = 1.82, SE = 0.80, p = .023) and enacted sexual orientation stigma (β = 1.33, SE = 0.11, p = .003) were also associated with greater anxiety symptoms. Additionally, individuals with high social support and high anticipated PrEP stigma had fewer depressive symptoms than individuals with low social support and high anticipated PrEP stigma (β = −3.25, SE = 1.40, p = .022). This relationship was marginally significant for anxiety (β = −2.54, SE = 1.31, p = .055). Sexual orientation and PrEP stigma may affect PrEP users' mental health. Interventions addressing stigma are needed to support the uptake and sustained PrEP use in this population.