Up to now, the response of antibiotics upon ionizing radiation has been very scarcely reported. Here, we present the results of X-ray photoabsorption experiments on isolated rifamycin, a broad-range antibiotic against Gram-positive and Gram-negative bacteria. A mass spectrometer has been coupled to a synchrotron beamline to analyze cationic products of photoabsorption on protonated rifamycin dimer and monomer sodium salts. Absorption of a single photon in the 100–300eV energy range leads to ionization of the molecular system, followed by vibrational energy deposition and subsequent inter- and/or intramolecular fragmentation. Interestingly, we observe a proton transfer from sodiated rifamycin to rifamycin, a widely observed process in ionized molecular systems in the gas phase. Moreover, we show that another charge-transfer process occurs in both dimer and monomer: intramolecular sodium transfer, which has not been reported yet, to the best of our knowledge.