Despite extensive preclinical imaging with radiotracers developed by continuous-flow microfluidics, a positron emission tomographic (PET) radiopharmaceutical has not been reported for human imaging studies by this technology. The goal of this study was to validate the synthesis of the tau radiopharmaceutical 7-(6-fluoropyridin-3-yl)-5H-pyrido[4,3-b]indole ([18F]T807) and perform first-in-human PET scanning enabled by microfluidic flow chemistry. [18F]T807 was synthesized by our modified one-step method and adapted to suit a commercial microfluidic flow chemistry module. For this proof of concept, the flow system was integrated to a GE Tracerlab FXFN unit for high-performance liquid chromatography purification and formulation. Three consecutive productions of [18F]T807 were conducted to validate this radiopharmaceutical. Uncorrected radiochemical yields of 17 ± 1% of crude [18F]T807 (≈ 500 mCi, radiochemical purity 95%) were obtained from the microfluidic device. The crude material was then purified, and > 100 mCi of the final product was obtained in an overall uncorrected radiochemical yield of 5 ± 1% (n = 3), relative to starting [18F]fluoride (end of bombardment), with high radiochemical purity (≥ 99%) and high specific activities (6 Ci/μmol) in 100 minutes. A clinical research study was carried out with [18F]T807, representing the first reported human imaging study with a radiopharmaceutical prepared by this technology.