Molecular docking studies have been performed to assess antimicrobial potential of three 1,3,4-Thiadiazole derivatives containing azulene rings. The simulations were conducted on Mycobacterium tuberculosis DNA gyrase reaction core (PDB ID 3M4I), Staphylococcus aureus DNA gyrase (PDB ID 4P8O) and Escherichia coli DNA adenine methylase (PDB ID 4RTO). The relationships between the structures of compounds and their potential antimicrobial activity were investigated. Interactions with proteins fragment amino acids residues form the active binding site were elucidated and the results of docking are reported in terms of docking score, compared with the natural ligand behaviour. Better docking scores are obtained for all the investigated compounds than for the natural ligand, the co-crystalized (4S)-2-methyl-2,4-pentanediol, in the case of the Mycobacterium tuberculosis DNA gyrase. Two of the studied ligands present better binding affinities against Escherichia coli DNA Adenine Methyltransferase (DAM) than the natural ligand (Sinefungin). Regarding S. aureus gyrase, the investigated thiadiazole derivatives exhibit lower docking scores and less interactions than the natural aminobenzimidazole urea inhibitor. Our study can be useful to screen and design new active compounds possessing similar structural moieties.