Nineteen N'-(benzylidene)-2-[(pyrimidin-2-yl)thio]acetohydrazides were obtained via the nucleophilic addition-elimination reaction of 2-[(pyrimidin-2-yl)thio)]acetohydrazide with aromatic aldehydes. The synthesized compounds were tested in vitro against various bacteria by microdilution. Among these compounds, N'-(3-hydroxybenzylidene)-2-[(pyrimidin-2-yl)thio]acetohydrazide (3l) was found to be the most potent derivative (MIC 7.8-15.62 mu g/mL) against Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 12228), and Escherichia coli (O157:H7-Clinical isolate), respectively. This outcome confirms that m-hydroxyphenyl moiety may have a considerable influence on antibacterial activity of the series tested. In the view of the results of the current work, further research can be carried out on the development of new effective antibacterial agents by the modification of compound 3l.