The photophysics and photochemistry of nalidixic acid (NA) were studied as function of pH and solvent properties. The ground state of NA exhibits different protonated forms in the range of pH 1.8–10.0. Fluorescence studies showed that the same species exist at the lowest singlet excited state. Absorption experiments were carried out with NA and with the methylated analog of nalidixic acid (MNE) in different organic solvents and water pH 3, where the main species corresponds to that protonated at the carboxylic group. These studies and the DFT calculation of torsional potential energy profiles suggest that the most stable conformation of the NA in nonprotic solvents corresponds to a closed structure caused by the existence of intramolecular hydrogen bond. Absorption and fluorescence spectra were studied in sulfuric acid solution. The pK value (H_o −1.0) found in these conditions was attributed to the protonation of the 4′ keto oxygen atom of the heterocyclic ring. Theoretical calculations (DFT/B3LYP/6-311G*) of the energies of the different monoprotonated forms of the NA and Fukui indexes (f_x⁻) showed that the species with the proton attached to 4′ keto oxygen atom is the most stable of all the cationic forms. MNE and enoxacin also showed the protonation of the 4′ keto oxygen atom with similar pK values. The photodecomposition of NA is dependent on the medium properties. Faster decomposition rates were obtained in strong acid solution. In nonprotic solvents, a very slow decomposition rate was observed.