The nitrocatechol derivatives tolcapone (1) and entacapone (2), used as adjunctive therapy in the treatment of Parkinson's disease, were investigated for their potential to inhibit the tau-derived-hexapeptide 306VQIVYK311. They were compared to small molecules that contain similar pharmacophores including the catechol derivatives (dopamine 3 and epinephrine 4), nitroderivatives (nifedipine 5 and chloramphenicol 6), nitrocatechol isomers (7 and 8), and a tolcapone derivative (13) lacking the nitrocatechol moiety. The aggregation kinetics by thioflavin S fluorescence assay indicates that both tolcapone (1) and entacapone (2) exhibit antiaggregation properties. These findings were supported by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy measurements which suggest that the nitrocatechol (3,4-dihydroxy-5-nitrophenyl) moiety is a suitable pharmacophore in the design of new tau-aggregation inhibitors. Furthermore, tolcapone (1) was identified as most active compound with antiaggregation activity (46% inhibition of fluorescence intensity at 50 μM), which was supported by TEM data. The in silico steric zipper model of the tau-derived-hexapeptide 306VQIVYK311 indicates that the 3,4-dihydroxy-substituent present in tolcapone (1) and entacapone (2) underwent polar contacts with lysine side chains (VQIVYK), whereas the charged 5-nitrosubstituent was in close contact with lysine side chain present in the steric zipper region suggesting the critical role of a nitrocatechol (3,4-dihydroxy-5-nitrophenyl) pharmacophore present in tolcapone (1) and entacapone (2) in tau-hexapeptide binding and prevention of β-sheet assembly. Our results have significant implications in the design and development of tau-aggregation inhibitors.