Mycobacterium tuberculosis (MTB), the pathogen responsible for tuberculosis (TB), remains a significant global health concern, especially with the growing prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. This study focuses on understanding the molecular basis of pyrazinamide (PZA) resistance, particularly mutations in the pyrazinamidase (Pzase) enzyme, including D8G, H71R, K96T, and S104R. We used computational methods to explore the effects of bioactive compounds on these PZA-resistant mutations. The structures of wild-type (WT) Pzase and its mutant variants were prepared, and molecular docking simulations were carried out using the CDOCKER protocol to assess potential binding interactions. To evaluate the stability of these interactions, we performed 0.5 μs molecular dynamics (MD) simulations followed by MM-PBSA analysis to calculate the binding free energies. Our results showed that garcinone D and neodiospyrin had stronger binding affinities than the reference molecule, pyrazinoic acid (POA), across both WT and mutant forms of Pzase. These compounds demonstrated lower Root Mean Square Deviation (RMSD) and radius of gyration (Rg) values, suggesting more stable binding interactions. Further validation through steered molecular dynamics (SMD) simulations indicated that garcinone D and neodiospyrin required significantly higher pulling forces to dissociate from the binding site compared to POA. Additionally, umbrella sampling simulations revealed more negative binding free energies for these two compounds, reinforcing their strong interaction with Pzase. These findings position garcinone D and neodiospyrin as promising candidates for the development of new treatments for MDR-TB and XDR-TB, offering a potential strategy to combat drug-resistant TB. This study provides valuable insights into the binding mechanisms and stability of these compounds, advancing the search for novel anti-TB therapies.