Heterocyclic compounds containing thiazolo and pyrimidine rings have attracted significant interest due to their diverse biological activities and structural versatility. The fusion of thiazole with pyrimidine or pyridine frameworks enhances their pharmacological potential and electronic stability. This work aimed to synthesize new heterocyclic derivatives based on benzylidene-thiazolidin-4-one scaffolds and evaluate their electronic, structural, and biological characteristics using both experimental and computational approaches. Benzylidene-thiazolidin-4-one derivatives 1 and 2 were condensed with guanidine hydrochloride to afford thiazolo[4,5-d]pyrimidin-5-amine and methyl benzoate 4. Subsequent reactions with malononitrile yielded thiazolo[4,5-d]pyrimidin-5(6H)-thione (5) and thiazolo[4,5-b]pyridine-6-carbonitrile 6. Density Functional Theory (DFT) calculations were performed to predict the molecular geometry, dipole moments, bond angles, bond lengths, and electronic properties. Reactivity and stability were evaluated from the HOMO–LUMO energy gap. Molecular docking studies were conducted to assess binding affinity with antibacterial and antimalarial protein targets. DFT analysis indicated that compound 3 exhibited the highest softness (σ =16.129 au), whereas compound 6 behaved as a hard compound (σ =18.519 au). HOMO electron density in compounds 3 and 5 was mainly localized on the NO2 group (98.4–100%), while in compounds 4 and 6, it was delocalized across the dihydrothiazolo and aromatic rings. Excited states of compound (3) were assigned to π–π* transitions (λ =208.09–223.42 nm) and n–π* transitions (λ =255.38–324.46 nm). Molecular docking revealed strong ligand–protein interactions, including four conventional hydrogen bonds, confirming high stability and binding affinity toward antibacterial and antimalarial targets. The synthesized thiazolo[4,5-d] and thiazolo[4,5-b] derivatives demonstrated favorable electronic properties and strong protein binding affinities, suggesting their potential as promising scaffolds for antimicrobial and antimalarial drug development.