Abstract : Tuberculosis (TB) remains a global health concern, particularly in developing countries, with Mycobacterium tuberculosis (M. tb) as its main causative agent. The 6 kDa Early Secreted Antigenic Target (ESAT-6) protein serves a function in M. tb virulence by disrupting phagosomal membranes and evading immune detection. This study employs in silico techniques to analyze the physicochemical properties, structural characteristics, and binding interactions of ESAT-6 with major histocompatibility complex (MHC) class II HLA-DR subunits. Rendering the model was done using SWISS-MODEL integrated with model validity parameters (i.e. QMEAN, Ramachandran Plot Analysis and MolProbity) which exhibited results proving the rendered model was reliable though with a few discrepancies. ESAT-6 exhibited hydrophilic and hydrophobic sides and aliphatic molecules in its core that influenced its functional properties. Physicochemical properties and secondary structures were characterized using ProtParam, ProtScale, and NetSurfP 3.0. Moreover, it was found that the majority of the protein has >25% relative surface area (RSA) with its N-terminal and C-terminal regions being the most exposed. Using ClusPro and PRODIGY for molecular docking, HLA-DRB3 exhibited the highest affinity to ESAT-6 (ΔG = -11.4 kcal/mol; Kd = 9.4×10-9), suggesting a role in the host’s immune response. The apolar-apolar interfacial contacts (ICs) contributed to most of the binding energy with evidence of consistent binding motifs between ESAT-6 and the HLA-DRB3.