Matrix metaloproteinase (MMP)-9, as a neuroinflammation-facilitating element, plays a significant role in neurodegenerative disorders and could be considered an inflammation control target. This study is intended to survey the effects of thioridazine (TRZ) and phenothiazines (PRZ) as neuroleptic drugs on the performance of recombinant human MMP-9 (rhMMP-9) by using experimental (ultraviolet visible (UV–vis), fluorescence, synchronous fluorescence, fluorescence resonance energy transfer (FRET) spectroscopy, circular dichroism (CD), surface plasmon resonance (SPR), and theoretical techniques (molecular docking). For this purpose, rhMMP-9 was expressed and purified by utilizing recombinant E. coli bacteria. The kinetic analysis illustrated that the different concentrations of TRZ and PRZ could considerably decrease the catalytic function of rhMMP-9 through competitive and mixed inhibition mechanisms, respectively. The results illustrated the higher affinity of TRZ-MMP-9 with an inhibitory IC50 value of 10.7 µM in comparison with PRZ (12.1 µM) following the conformational alteration of the enzyme. In addition, hydrophobic binding and hydrogen bonds/van der Waals forces were suggested for TRZ-MMP-9 and PRZ-MMP-9, respectively. Finally, the molecular docking study demonstrated that TRZ and PRZ bind to the cavity in the catalytic and fibronectin domains, respectively, which supports the experimental data. This study provides some experimental and structural insights into the possible anti-inflammatory mechanisms of TRZ and PRZ drugs that suggest both drugs could be used as anti-MMP-9 drugs in neuroinflammatory conditions.