Catalase as an important antioxidative enzyme protects cells from the toxic effects of hydrogen peroxide. 2-hydroxy-1,4-naphtoquinone, as a naphthoquinone derivative, has been found to have a broad biological and pharmacological activities, including anti-cancer and anti-bacterial effects. In this study, the effect of two different derivatives of 2-hydroxy-1,4-naphtoquinone derivatives (BNQ and ANQ) on the structure and function of bovine liver catalase (BLC), was studied using different spectroscopic and computational methods such as UV-vis spectrometry, fluorescence and FTIR spectroscopy, and molecular docking studies. Kinetic studies and thermodynamic parameters showed that by adding gradual concentrations of BNQ and ANQ, catalase activity was significantly decreased through mixed and competitive inhibition mechanisms, respectively. The fluorescence spectroscopic results at different temperatures indicated that BNQ and ANQ can quench the intrinsic fluorescence emission of BLC through static quenching mechanism. Molecular docking data in agreement with experimental results, confirmed that there is only one binding site on the BLC structure for BNQ and ANQ. Study on effect of different compounds on the activity and structure of the catalase as a bilateral actions and principal biological enzyme could be important. Because excessive utilization of these compounds in various drugs can cause side effects and may threaten human health.