Summary We carried out hydrogeological, geophysical, hydrochemical, isotopic, and molecular biological investigations in a fractured carbonate aquifer in southern Italy to verify if prolonged variations in groundwater salinity with depth can be due to mixing processes between fresh infiltration water and groundwater. All investigations revealed the formation of a non-permanent halocline at the experimental site, whose thickness and shape varies over time. Variations in thickness and shape are influenced by infiltration processes. Three main types of Electrical Conductivity (EC) profiles were found during the research period. In the high-flow period, the EC profile consisted of a transition layer and a nearly homogeneous higher salinity groundwater layer, and no mixed layer was observed. A nearly-homogeneous mixed layer was detected in the low-flow period, excluding the late recession when the EC profile was characterized by a higher salinity groundwater layer and a negligible transition layer. The EC profile through the transition layer was approximately symmetric and linear, probably due to a velocity shear across this layer. The velocity shear was probably caused by the difference in opening-porosity detected between the upper and lower carbonate bedrock through geophysical investigations. The investigated phenomenon is due to water that infiltrates very close to the observation well. In fact: (a) the isotopic composition of the lower salinity groundwater layer in early recharge 2007/2008 (δ 18O = −8.12‰; δ 2H = −49.92‰) is very close to the composition of rainwater collected in the same period near the observation well (δ 18O = −8.19‰; δ 2H = −51.35‰); (b) the C DIC composition in the lower salinity groundwater layer (−15,96‰) is very close to the composition (−15.77‰) in the infiltration water collected at the bottom of the soil medium, close to the observation well; (c) the lower salinity groundwater layer is characterized by significant concentrations of acetate (up to 4.5 mg/L) as well as the saturated paste extracts obtained only from the soil medium collected close to observation well (up to 110 mg/L); (d) in the lower salinity groundwater layer acetate-assimilating bacteria belonging to Betaproteobacteria and Bacteroidetes were found, further supporting that this groundwater layer significantly interacts with the soil medium described above. Similar mean 3H contents in local rainwater (4.6 TU), in the lower salinity groundwater layer (4.5 TU), in the higher salinity groundwater layer (4.5 TU) and in spring water (4.4 TU) clearly show that the halocline formation is not influenced by differences in residence times.