•Hydraulic diffusivity estimated using analytical equations agree with regional model.•Strongest tidal fluctuation in high permeability zone of Avon Park Formation.•Tidal springs are important sea water/groundwater interfaces.•Preferential transmission of tidal wave in subsurface conduits. Interaction between ocean tides and coastal aquifers is an important process influencing the quality and quantity of groundwater resources. In this study, high frequency groundwater fluctuation along two transects from the Gulf of Mexico to the Upper Floridan Aquifer (UFA) were analyzed to characterize the hydrogeologic structure of the multi-layer karst aquifer. Tidal efficiency and time lag of harmonic constants were calculated from groundwater and seawater level and aquifer diffusivity were estimated using the Jacob-Ferris equation. Measurable tidal signals were observed at a maximum inland distance of approximately 7–8 km due to the high conductivity of the unconfined karst aquifer. The largest tidal fluctuation was observed in the deeper unit of the aquifer (146–155 m below land surface), which indicate the hydraulic contact between seawater and the high permeability zone of Avon Park Formation. The estimated values of hydraulic diffusivities agreed well with the regional groundwater flow model despite the highly heterogeneous nature of karst aquifer. Our analysis suggested that tidal springs are important sea water/groundwater interfaces and the conduit networks of tidal creeks, caves, and fractures are preferential pathways of tidal propagation.