In light of the growing concern surrounding antimicrobial resistance (AMR), there is an urgent need for novel antimicrobial agents with enhanced efficacy and reduced susceptibility to resistance mechanisms. Heterocyclic compounds, particularly those incorporating sulfur and oxygen heteroatoms, have emerged as promising candidates due to their diverse pharmacological properties and potential to overcome resistance mechanisms. In the present study, a novel class of thiadiazole derivatives were amalgamated having functionalities of benzimidazole and pyridine ring structures. Spectral techniques including proton NMR, 13C NMR, FTIR, mass spectroscopy, and elemental characterization were used to ascertain the molecular structure. The antibacterial and antifungal activity of the derivatives was assessed using the disc diffusion technique against selected bacterial and fungal strains. The derivatives showed significant antimicrobial activity compared to the standard drugs with MIC in the range of 25–100 μg/ml. The outcome of the structure-function analysis showed that para-positional substitution of an electronegative substituent at the phenyl ring enhances antimicrobial activity. Molecular dynamic simulations study was conducted against the receptor protein dihydrofolate reductase (DHFR). The compounds 3c and 3e demonstrated the highest binding energies against the selected target. The correlation between experimental and computational results was found to be robust. Moreover, the chloro and nitro group substituent were identified as the key contributors to the enhanced antimicrobial activity of the derivatives. Additionally, the thiadiazole derivatives demonstrated promising ADMET profile, excellent oral bioavailability, and favorable drug-likeness, and were also found to be “Lipinski's rule of five compliant”.