The on-surface synthesis of nonacene has been accomplished by dehydrogenation of an air-stable partially saturated precursor, which could be aromatized by using a combined scanning tunneling and atomic force microscope (STM/AFM) as well as by on-surface annealing. This transformation allowed the in-detail analysis of the electronic properties of nonacene molecules physisorbed on Au(111) by scanning tunneling spectroscopy (STS) measurements. The spatial mapping of molecular orbitals was corroborated by density functional theory (DFT) calculations. Furthermore, the thermally-induced dehydrogenation uncovered the isomerization of intermediate dihydrononacene species, which allowed for their in-depth structural and electronic characterization.