Navigated transcranial magnetic stimulation (nTMS) is a painless method for targeting stimulation of the human brain. The responses from peripheral muscles provide a direct measure for the integrity of the cortical interneurons, corticospinal neurons, and spinal motoneurons. Parkinson's disease (PD) is characterized by the degeneration of dopaminergic nigrostriatal pathways and by the lateralization of motor dysfunction. In this study, we applied nTMS on a cohort of PD patients and healthy subjects (HC) in order to investigate the asymmetry of the cortical excitability. During the experiments, resting motor threshold (rMT) in each hemisphere and its difference between brain sides (ΔrMT), motor evoked potentials (MEPs) amplitude, and the electric field strength at the optimal stimulus location (E-field) were evaluated for each subject. A statistical analysis was performed and a significant difference between HC and PD was found in resting motor threshold asymmetry descripted by ΔrMT. This finding suggested that ΔrMT could be considered as an informative biomarker of PD disease. The innovative approach of navigated magnetic stimulation procedure allowed the respect of the cortical architecture through the accurate spatial location. Indeed, no significant differences were found in E-field strength in both hemispheres. The optimal spatial specificity of navigated TMS provides support for its application in the neurodegenerative disease scenario.