Structural integrity of the bacterial genome plays an important role in bacterial survival. Cellular consequences of an intolerable amount of change in the DNA structure are not well understood in bacteria. Here we have stated that binding of synthetic 6-nitroquinoxaline derivatives with DNA led to change in its global structure, subsequently culminating with over-supercoiled form through in-path intermediates. This structural change results in induction of programmed cell death like physiological hallmarks, which is dependent on substitution driven structural modulation properties of the scaffold. A sublethal dose of a representative derivative, 3a, significantly inhibits DNA synthesis, produces fragmented nucleoids, and alters membrane architecture. We have also shown that exposure to the compound changes the native morphology of Staphylococcus aureus cells and significantly disrupts preformed biofilms. Thus, our study gives new insight into bacterial responses to local or global DNA structural changes induced by 6-nitroquinoxaline small molecules.