Myocyte enhancer factor 2 (MEF2; MEF2A-MEF2D) transcription factors regulate gene expression in a variety of developmental processes by binding to AT-rich DNA motifs via highly conserved N-terminal extensions known as MADS-box and MEF2 domains. Despite the fact that MEF2 proteins exhibit high similarity at their N-terminal regions and share a common consensus DNA binding motif, their functional preferences may vary significantly in the adjacent regions to the DNA binding core segment. The current study delineates the conformational paradigm, clustered recognition, and comparative DNA binding preferences for MEF2A and MEF2B-specific MADS-box/MEF2 domains at the YTA(A/T)4TAR consensus motif. In both MEF2A and MEF2B proteins, α1-helix plays a crucial role through acquiring more flexibility by attaining loop conformation. In comparison to apo-MEF2, an outward disposition of the distal portion of α1-helix and movement of its proximal part to β1 allows synergistic repositioning of the α1-α2 linker, C-terminal region, and MEF2 domain, resulting in the formation of a hydrophobic groove for DNA binding. In both instances, conformational switching of the helical content is the main contributing factor while preserving the overall β-topology to maintain the inside-out conformation of subdivided α1-helix flip. Multivariate statistical analysis reveals that MEF2B obscures less accessible conformational space for DNA binding as compared to the MEF2A-DNA complex. The presence of similar structural requirements and conserved residues including Arg10, Phe21, and Arg24 in accentuating the MEF2-specific DNA recognition mechanism led us to perform structure-based virtual screening for isolating novel inhibitors that are able to target MEF2-DNA binding regions. The top hits (acetamide, benzamide, carboxamide, and enamide) obtained through preliminary assay were scrutinized to binding potential analysis at the MEF2-DNA binding groove, energy values, absorption, distribution, toxicity, and Lipinski's rule of five assessments. Based on these findings, we propose valuable active drug-like molecules for selective applications against MEF2A and MEF2B. The current study may help in uncovering the atomistic-level mechanistic DNA binding patterns of MEF2 proteins, and data may be valuable in devising effective therapeutic strategies for MEF2-associated disorders.