Hydrothermal systems are often dichotomous with respect to their chemistry and microbial diversity. One model explaining this dichotomy is phase separation due to decompression boiling of hydrothermal fluid as it ascends through the subsurface. Phase separation results in two types of fluids: alkaline-chloride waters, representing boiled residual liquid, and acid-sulfate waters, representing condensate of released vapor. While phase separation helps to explain first-order patterns in the chemistry and biology of a hot spring’s surficial expression, our understanding of these systems is only two-dimensional. The architecture and timescales of subsurface fluid flow remain unknown. By combining geophysical, geochemical, isotopic and biological measurements from two adjacent, phase-separated pools, we show that phase-separated fluids preserve distinct characteristics from two different regions in the Yellowstone hydrological regime, and that reactive transport, occurring over two different timescales, and shallow meteoric recharge are essential to establishing the geobiological feedbacks that drive geochemical and microbial diversity.