Candida albicans, an opportunistic pathogen, uptakes host-derived carbon sources for its survival and causes candidiasis infection. However, host defences such as macrophages restrict the C. albicans’ sugar uptake via phagocytosis, triggering the glyoxylate pathway for adaptation in nutrient-limited conditions. Within macrophages, the C. albicans malate synthase (CaMLS1) exploits the available acetyl-CoA (ACOA), which condenses in presence of Mg 2+ with glyoxylate (GOXL) to form malate, supporting its survival. The absence of structural studies highlights the need for in silico interventions to better understand CaMLS1 functional architecture thereby aiding in design of the therapeutic drug agents. In this study, computational approaches such as MD simulations, correlations studies and binding affinity analysis were employed to delineate the structural dynamics and to investigate the interactions of cofactors and substrates with CaMLS1. From the results, template-based 3D modeling revealed that CaMLS1 has three domains: N-terminal, TIM-barrel and C-terminal domain, of which TIM-barrel found to be dynamic through MD studies. However, substrates (GOXL and ACOA) binding to CaMLS1 markedly reduced this dynamism, indicating the stability of the complex. The binding free energy calculations of the complex showed that ACOA binds with notably higher affinity (-921 kJ/mol) than GOXL (-6 kJ/mol), consistent with previous observations reported in literature. Furthermore, presence of cofactors and substrates modulated the tunnel availability at the active site, thereby affecting substrate’s access and product release through changes in the enzyme’s conformational dynamics. The findings from the structure and dynamics studies of CaMLS1, as well as the key residues involved in substrate binding offer scope for design of molecules targeting this enzyme for antifungal therapy.