Bacterial infections are a global issue, causing sickness and death, mainly in developing nations. Resistance to current medicines is a significant issue in healthcare. Overcoming the resistance problem will necessitate the development of molecules with novel modes of action that do not cross-react with existing medicines. Synthesis of new bis-heterocycles linked to the piperazine core and vanillin unit is disclosed in this regard. The target compounds were produced via the reaction of 4,4′-((piperazine-1,4-diylbis(2-oxoethane-2,1-diyl))bis(oxy))bis(3-methoxybenzaldehyde) with various reagents using the Hantzsch, Biginelli, and Michael’s reactions. All the generated compounds were tested for their antibacterial activity against a variety of bacterial strains. The antibacterial activity evaluations revealed that compound 32 has the most promising activity against Pseudomonas aeruginosa (19.5 ± 0.7 mm) compared to Ofloxacin (17 mm) with minimum inhibitory concentration (MIC) equaled to 625 µg/mL. A molecular docking investigation revealed that compound 32 had the greatest binding affinity for bacterial enoyl reductase (−36.13 ± 0.2 Kcal/mole) when compared to the co-crystallized ligand (−16.8 ± 0 Kcal/mole).