Travertine deposits preserved within ancient aqueduct channels record information about the hydrology, temperature, and chemistry of the flowing water from which they precipitated. However, travertine is also chemically reactive and susceptible to freshwater diagenesis, which can alter its original composition and impact reconstructions of aqueduct operation, maintenance, and climate. Hydraulic reconstructions, in combination with a suite of high-resolution optical, laser, electron, and X-ray microscopy analyses, have been used to determine the original crystalline structure and diagenetic alteration of travertine deposited in the Anio Novus aqueduct built in A.D. 38–52 at Roma Vecchia. Age-equivalent travertine deposits, precipitated directly on the mortar-covered floor at upstream and downstream sites along a 140-m-long continuous section of the Anio Novus channel, exhibit consistent crystalline textures and stratigraphic layering. This includes aggrading, prograding, and retrograding sets of travertine linguoid, sinuous, and hummocky crystal growth ripples, as well as sand lags with coated siliciclastic grains deposited on the lee slope of ripple crests. The original aqueduct travertine, which is similar to travertine formed in analogous natural environments, is composed of shrub-like, dendritically branching aggregates of 1–3-μm-diameter euhedral calcite crystals. Dark brown organic matter-rich laminae, formed by microbial biofilms and plant debris, create stratigraphic sequences of high-frequency, dark–light layering. This hydraulic and petrographic evidence suggests that large, radiaxial calcites diagenetically replaced the original aqueduct travertine shrubs, forming upward-branching replacement crystals that crosscut the biofilm laminae. While this diagenetic process destroyed the original crystalline fabric of the calcite shrubs, the entombed biofilm laminae were mimetically preserved. These integrated approaches create the type of depositional and diagenetic framework required for future chemostratigraphic analyses of travertine deposited in the Anio Novus and other ancient water conveyance and storage systems around the world, from which ancient human activity and climatic change can be more accurately reconstructed.