Main objectives of this study were to investigate whether humic substances (HS) exist in hot groundwater from deep basinal aquifers waters and to provide basic knowledge on humic (HA) and fulvic acids (FA) isolated from these fluids. Water sampling and HS isolation was carried out in three consecutive years from wells tapping the aquifers of the Pannonian Basin (Southeast Hungary). The isolated humic acid and fulvic acid samples were studied by elemental analysis, Fourier transform infrared (FTIR) spectroscopy, and Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI-FT-ICR/MS). We found that modified sampling (acidification of water samples either immediately or after cooling down) had no traceable effect on the properties of isolated HAs and FAs. The quantity and quality of humic materials did not change in three years. The analytical results clarified distinctive chemical characters of HS in the deep aquifer groundwater compared to the Suwannee river fulvic acid (SRFA) as a reference. In addition, we stated that aliphatic content of isolated HAs and FAs increased with increasing aquifer depth and temperature, but the aromaticity and heteroatom content decreased in both fractions.