A family of 11 nitrophenol 2‐nitro‐5‐(4‐substituted phenylquinolin‐2‐yl)phenol derivatives (4, 4a‐j) was effectively synthesized as antimicrobial medications. A mixture of the substituted 3‐hydroxy‐4‐nitrobenzaldehyde substituted aromatic amine and substituted phenylacetylenes were used to synthesis the title compounds 4, 4a‐j. Antimicrobialactivity potential of 4, 4a‐j was evaluated against Streptococcus pyogenes (MTCC 442), Staphylococcus aureus (MTCC 96), Pseudomonas aeruginosa (MTCC 424), and Escherichia coli (MTCC 443). DNA gyrase inhibition studies carried out to understand the mechanism ofaction of the antimicrobial effect of target compounds. HRBC membrane stabilization (in vitro) property was also assessed as a representative human cellular cytotoxic effect of 4, 4a‐j since HRBC alike lysosomal cells and the lysozyme activity leads to inflammation and its adverse effects in cellular systems. Results reveal that compounds 4c and 4h have remarkable antibacterial activity and screened for further preclinical studies.