Background: The prevalence of human T-lymphotropic virus type-1 (HTLV-1) infection is higher in women, and sexual intercourse has been described as an important route of male-to-female transmission. The present study aimed to quantify HTLV-1 proviral load (PVL) in vaginal fluid, and to investigate correlations with PVL in peripheral blood mononuclear cells (PBMCs). In addition, cytopathological alterations and vaginal microbiota were evaluated. Methods: HTLV-1-infected women were consecutively recruited at a multidisciplinary center for HTLV patients in Salvador, Brazil. All women underwent gynecological examinations to obtain cervicovaginal fluid and venipuncture for blood collection. PVL, as measured by real-time quantitative polymerase chain reaction (RT–qPCR), was expressed as the number of copies of HTLV-1/106cells in blood and vaginal fluid samples. Light microscopy was used to assess cervicovaginal cytopathology and vaginal microbiota. Results: In the 56 included women (43 asymptomatic carriers and 13 diagnosed with HTLV-1-associated myelopathy/tropical spastic paraparesis—HAM/TSP), mean age was 35.9 (SD ± 7.2) years. PVL was higher in PBMCs (median: 23,264 copies/106cells; IQR: 6776–60,036) than in vaginal fluid (451.9 copies/106cells; IQR: 0–2490) (p< 0.0001). PVL in PBMCs was observed to correlate directly with PVL in vaginal fluid (R = 0.37,p= 0.006). PVL was detected in the vaginal fluid of 24 of 43 (55.8%) asymptomatic women compared to 12 of 13 (92.3%) HAM/TSP patients,p= 0.02. Cytopathologic analyses revealed no differences between women with detectable or undetectable PVL. Conclusion: HTLV-1 proviral load is detectable in vaginal fluid and correlates directly with proviral load in peripheral blood. This finding suggests that sexual transmission of HTLV-1 from females to males may occur, as well as vertical transmission, particularly in the context of vaginal delivery.