The current work focuses on the synthesis and characterization of three Schiff bases synthesized under microwave conditions from 5-nitrothiophene-2-carboxaldehyde and substituted aromatic amines. These compounds were successfully synthesized with high yields, and they were examined using FTIR, NMR (1H), and mass spectroscopic techniques. Density functional theory (DFT) was used to theoretically examine the quantum chemical characteristics of synthesized compounds employing the B3LYP/6-31G(d,p) method. These substances were also tested for antibacterial activity. Few of the microorganisms under investigation were found to be resistant to these compounds. Chemical reactivity characteristics and molecular docking investigations have been linked to the antibacterial properties. Additionally, using the electronic absorption titration method, the compounds' DNA-binding capabilities have been studied experimentally. The outcomes are very encouraging, so we can say that these compounds are biologically less active and can be used with structural modifications towards drug development.