Electronic absorption and fluorescence spectra of ciprofloxacin, norfloxacin, enoxacin, pefloxacin and nalidixic acid in aqueous solutions were investigated. The time-resolved fluorescence spectra were measured and interpreted. The changes of the luminescence spectra and electron structure of the compounds under study are explained by different degrees of the spin-orbital interaction caused by nitrogen heteroatoms lone pairs effect. Possible ways of the protonation process for naphtyridine and quinolone rings with different substitutions are discussed. The photophysical behavior of FQs was studied using density functional theory (DFT) calculations.