New 3-hydrazinecarbothioamide-N-benzylindol derivatives 3a/b have been synthesized by the reaction of N-benzyl-3-acetylindol 1 with hydrazinecarbothioamide 2a/b. Then compounds 3a/b were cyclized with different α-halocarbonyl derivatives to produce thiazolidinone and thiazole compounds in moderate to excellent yields. The anti-microbial activities were screened for all the newly synthesized compounds. The experimental results indicated that only hydrazinecarbothioamides 3a,b showed good anti-microbial activities toward all the studied microbes compared with the used references. Despite that, compound 12e had anti-bacterial efficacy versus E. coli, compound 12d had anti-microbial activity against C. albicans, and compounds 12c and 12d had anti-fungal action against A. niger. Also, compounds 3a and 3b exhibited higher MIC values than ciprofloxacin. A molecular docking study was conducted for the most potent compounds 3a/b to assess theoretically the anti-microbial effect on the crystal structures of S. aureus and E. coli DNA gyrase B enzymes. The results of molecular docking proved that hydrazinecarbothioamides 3a,b had good binding energy and good binding mode actions towards the DNA gyrase B enzymes through their action with the main amino acids of the aimed enzymes in a similar way to the native inhibitor, consequently preventing cell division that leads to the death of bacteria.