We report the characterization of five strains belonging to the halotolerant highly related Debaryomyces hansenii/fabryi species. The analysis performed consisted in studying tolerance properties, membrane characteristics, and cation incell amounts. We have specifically investigated (1) tolerance to different chemicals, (2) tolerance to osmotic and salt stress, (3) tolerance and response to oxidative stress, (4) reactive oxygen species (ROS) content, (5) relative membrane potential, (6) cell volume, (7) K+ and Na+ ion content, and (8) membrane fluidity. Unexpectedly, no direct relationship was found between one particular strain, Na+ content and its tolerance to NaCl or between its ROS content and its tolerance to H2O2. Results show that, although in general, human origin D. fabryi strains were more resistant to oxidative stress and presented shorter doubling times and smaller cell volume than food isolated D. hansenii ones, strains belonging to the same species can be significantly different. Debaryomyces fabryi CBS1793 strain highlighted for its extremely tolerant behavior when exposed to the diverse stress factors studied.