The photophysical behavior of the quinolone antibiotics, oxolinic (OX), cinoxacin (CNX) and pipemidic (PM) acids was studied as a function of pH and solvent properties. The ground state of these compounds exhibits different protonated forms, which also exist in the first excited states. Theoretical calculations of the Fukui indexes allowed to assigning the different protonation equilibria. The pK values indicate that the acidity of the 3-carboxylic and 4-carbonyl groups increases with the N-atom at position 2 in CNX. It has been found that fluorescence properties are strongly affected by pH, the more fluorescent species is that with protonated carboxylic acid, protonated species at the carbonyl group and the totally deprotonated form present very low fluorescence. The fluorescence behavior also depends on the chemical structure of the quinolone and on the solvent properties. The analysis of the solvent effect on the maximum and the width of the fluorescence band of OX, using the linear solvent–energy relation solvatochromic equation, indicates that the polarizability and hydrogen bond donor ability are the parameters that condition the spectral changes. The hydrogen bond acceptor ability of the solvents also contributes to the spectral shifts of CNX. The compound bearing the piperazinyl group at the position 7, PM only is fluorescent in high protic solvents. These results are discussed in terms of the competition between the intra- and intermolecular hydrogen bonds. The irradiation of OX, CNX and PM using 300 nm UV light led to a very low photodecomposition rate. Under the same conditions the nalidixic acid (NA), a structurally related quinolone, photodecomposes two orders of magnitude faster.