A series of 10 p‐substitutedbenzoylmethylene hydrazide derivatives 4a‐j were synthesized by protecting carboxylic group of 4‐hydroxybenzoic acid using methanol and sulfuric acid than reacting it with hydrazide to form 4‐hydroxybenzohydrazide followed by reacting with a variety of aldehydes and evaluated for their activity against nosocomial infection. All the synthesized compounds were characterized by Fourier‐transform infrared (FT‐IR), 1H nuclear magnetic resonance (NMR), and mass spectral data. The in vitro antimicrobial potential of synthesized compounds was estimated against prominent strains of nosocomial pathogens (Staphylococcus aureus, Escherichia coli, and Aspergillus niger). The antimicrobial evaluation revealed compounds 4b, 4c, 4d, 4e, 4f, and 4j to be the most active compounds of the series with IC50 value for antibacterial in the range 0.39 to 0.75 μM/mL. Furthermore, the in vitro cytotoxic potential of the compounds was appraised by hemolytic assay. The results showed that some of the synthesized compounds exhibited marked activity.