While there have been great advances in HIV prevention methods in recent years, such as daily pre-exposure prophylaxis (PrEP) and combination antiretrovirals used as Treatment as Prevention (TaSP), access to care, compliance, and consistent follow-up with treatment remain barriers to care in high-risk populations. The purpose of this study was to explore the barriers and facilitators for the uptake and acceptability of PrEP among female sex workers in Tshwane District, South Africa. A descriptive qualitative research design was used. Female sex workers (FSW) aged 18 years and above were purposively sampled to participate in the study. The research team approached a minimum of 24 female sex workers until data saturation was achieved. The results showed that barriers such as a shortage of PrEP, side effects of PrEP, unawareness of collection points, reliance on pills, stigmatization, and absence of clinics influenced the uptake and acceptability of PrEP. The study also reported on facilitators that enhance the uptake and acceptability of PrEP among sex workers in Tshwane District. These facilitators include knowledge and awareness, perceived HIV risk, availability of mobile clinics, the effectiveness of PrEP, easier accessibility of information, awareness programmes, fear of infection, and preventative measure. This study established the barriers and facilitators that potentially influence the uptake and acceptability of PrEP among female sex workers in the Tshwane District, Gauteng Province, South Africa.