Abstract: The antibacterial efficacy of a Cu(II) complex of 2-hydroxyphenyl-azo-2 ′ -naphthol (HPAN) was studied on gram-positive Bacillus subtilis and gram-negative Escherichia coli. In vitro antimicrobial activity was determined by an agar-well diffusion assay and minimum inhibitory concentration. Cu II (HPAN) 2 was found to be better than HPAN in antibacterial activity. Although both bacterial strains succumbed to high concentrations of each compound, at low concentrations only Cu II (HPAN) 2 was active on B. subtilis. To explain the observations, reductive cleavage of the azo-bond to aromatic amines was followed by an in vitro enzyme assay using cell extracts of E. coli containing azo-reductase. Interaction of Cu II (HPAN) 2 with calf thymus DNA was compared with HPAN for correlation with antibacterial activity. Enzyme-assay on the reductive degradation of azo bond and DNA interaction do not individually explain trends observed in antibacterial activity. Comparable binding of Cu II (HPAN) 2 and HPAN with calf thymus DNA was attributed to the presence of anionic species of the complex in solution. Significant activity of the complex was probably due to effective cellular uptake of it by bacterial cells as shown by a fluorescence study. The activity of the complex resembled some established antimicrobial agents. Since the complex has a moiety, not common to most antibacterial agents, resistance towards it should be significantly less, hence an advantage. Graphical Abstract: Synopsis: 2-Hydroxyphenyl-azo-2 ′ -naphthol (HPAN) and its Cu II complex were screened for antibacterial activity by agar-well diffusion assay and minimum inhibitory concentration. Studies on azo bond cleavage by enzyme assay and DNA binding of the compounds were done to explain the antibacterial activity. The pronounced activity of Cu (II) -HPAN is attributed to an effective cellular uptake by bacterial cells. [Figure not available: see fulltext.]. © 2018, Indian Academy of Sciences.