We estimated the costs of Option B+ for HIV-infected pregnant women in 12 facilities in Morogoro Region, Tanzania, from a provider perspective. Costs of prevention of mother-to-child (PMTCT) HIV services were measured over 12 months to September 2017 to estimate the average costs per HIV testing episode, per HIV-positive case diagnosed, per patient-year on antiretroviral therapy (ART), and per neonatal HIV care. A one-way sensitivity analysis was undertaken to understand how staffing levels and other core resource inputs affected costs. The total number of HIV testing episodes was 25,593 with 279 HIV cases identified yielding a 1.1% positivity rate. The average cost per testing episode was US$5.49 (range US$2.13 to US$13.93), and the average cost per HIV case detected was US$503.29 (range US$230.61 to US$3330.38). The number of pregnant women initiated on ART was 278. The mean cost per patient-year on ART was US$159.89 (range US$100.91 to US$812.23). The average cost of neonatal HIV care was US$90.09 (range US$41.53 to US$180.26). PMTCT service costs varied widely across facilities due to variations in resource use, number of women testing, and HIV prevalence. The study provides further evidence against generalising cost estimates, and that budgeting and planning requires context specific cost information.