Tau, as a neuronal protein, has a crucial role in microtubule assembly and stabilization. In the current study, fluorescence spectroscopy, surface plasmon resonance (SPR), as well as molecular docking analysis were used to explore the influence of benzodiazepines, clonazepam (CZP), and diazepam (DZP), on tau protein structure. SPR thermodynamic parameters of drugs-protein complexes were calculated and the results showed that both complexes have negative ∆H and ∆S and positive ∆G values. According to these findings, the roles of hydrogen bonds and van der Waals forces are extremely substantial in the formation of a complex between DZP/CZP and tau protein. It was observed that the KSV values declined following increasing temperature, therefore the fluorescence quenching mechanism of tau-CZP/DZP was static quenching. Molecular docking studies represented that hydrogen bonds and hydrophobic interactions are important in the formation of complexes between CZP/DZP and tau protein. This study provides general and significant information about the structural behavior of tau protein following the binds to both benzodiazepine derivatives CZP and DZP. Also, this investigation led us to the idea that CZP and DZP could be effective compounds to prevent the aggregation of tau protein and the treatment of tauopathy.