Full-service sex workers (FSSWs) are at heightened risk of contracting HIV due to facing multi-level challenges to sexual health. This study investigated factors associated with willingness to use Pre-Exposure Prophylaxis (PrEP)—a daily HIV preventative medication, among FSSWs. Using social–ecological theory, an online survey was developed with initial guidance from a local sex worker advocacy organization to assess barriers and facilitators to PrEP uptake willingness. The survey was disseminated with the assistance of local and national sex work advocacy organizations. In our sample of FSSWs (n = 83), two barriers and two facilitators initially were associated with PrEP uptake. However, in adopting a more conservative analysis, only anticipating stigmatizing disapproval from others for using PrEP and providing others with PrEP knowledge maintained statistical significance. These two variables collectively explained nearly 30% of the variance in PrEP uptake willingness. Implications for both future research and clinical work with FSSWs are discussed.