As part of ongoing studies in developing new antimicrobials, a novel series of N-(1, 3-benzothiazol-2-yl)-2-[(2Z)-2-(substituted arylidene) hydrazinyl] acetamide (5a–j) was synthesized in order to determine their antimicrobial activities. The synthesized compounds were tested in vitro against two Gram-positive, one Gram-negative bacteria and two fungal strains in comparison with control drugs. Microbiological results showed that the synthesized compounds possessed a broad spectrum of antibacterial activity against the tested microorganisms. The compounds with a 4-dimethylamino group (5j) and 4-amino group (5i) on the aromatic ring possessing azomethine linkage showed better antimicrobial activity; almost similar to that of standard drugs thus they could be promising candidates for novel drugs. The novel heterocycles were characterized by elemental analyses and spectral features.