Chromatin is the complex of deoxyribonucleic acid (DNA) with proteins that exists in eukaryotic cell nuclei. Chromatin was extracted from livers of Wistar rats and subjected to a 248-nm excimer laser radiation, in doses of 0.5–3 MJ/m2. An UV excimer laser Iofan 1701, with 40-mJ dose/pulse and frequency of 30 Hz was used. The radiolysis of chromatin was analyzed by (1) 1H-NMR spectroscopy, (2) steady-state fluorescence, (3) time-resolved fluorescence, and (4) fluorescence resonance energy transfer (FRET) methods. The laser action on chromatin determines bigger values of the transverse relaxation time (T2), which indicates less bound water in the chromatin structure, therefore a more injured one. The chromatin intrinsic fluorescence decreases on laser action, proving the destruction of the chromatin protein structure. By the time-resolved fluorescence we established that the relative contribution of the excited state lifetime of bound ethidium bromide to chromatin DNA diminishes with the laser dose. This denotes single- and double-strand breaks produced in DNA structure. By the FRET method, the energy transfer efficiency and the distance between dansyl chloride and acridine orange coupled at chromatin were determined. The distance increases with laser action. The determination of the chromatin structure modification in an excimer laser field can be of real interest in medical applications.