Sr and Nd isotopic ratios along with (REE + Y) and other trace elements were measured in bedded travertines from three quarries at Tivoli Terme, Latium, aimed at deciphering their chemical and isotopic characteristics for the origin and evolution of fluids. Travertines, which were deposited between ∼81 and ∼54 ka BP, display high Sr and low REY contents along with REYPAAS patterns, supporting their fluid origin mainly from marine rocks, in particular the Upper Triassic Burano Evaporite Fm. This has been confirmed by the narrow ranges of δ87Sr and εNd values, close to those of that formation and other Mesozoic marine carbonates from central Italy. A sample of today's thermal water from Tivoli Terme provided a REYPAAS pattern and a δ87Sr value comparable with travertines, both features supporting that today's water is analog to past hydrothermal fluids. The overlap of the chemical and Sr–Nd isotopic compositions of coeval travertines and thermal waters from Tivoli and Acquasanta Terme, Marche, suggests a common fluid origin, ruling out any significant contribution from magmatic fluids/rocks to Tivoli travertines, yet deposited at the periphery of the Alban Hills volcano. Lastly, the geochemical results provide implications for regional neotectonics and the role of CO2-rich fluid pressure in fault activation, even in mildly active to almost inactive tectonic domains.