By performing single molecule stretching experiments with optical tweezers, we have studied the DNA interaction with the ligand Hoechst 33258. The mechanical properties of the complexes formed as a function of ligand concentration were directly determined from these measurements by fitting the force × extension curve to the WormLike Chain model of semiflexible polymers. In addition, the physicochemical parameters of the interaction were extracted from the persistence length data by using a previously developed two-sites quenched disorder statistical model, allowing the determination of the binding isotherm. Such approach has allowed us to decouple the two different binding modes present in this system. In particular, it was found that the binding isotherm consists of two Hill-type processes, one noncooperative and the other strongly cooperative. Finally, DNA condensation due to the interaction with the ligand was also verified and characterized here by analyzing the apparent contour length of the complexes.