In current research, five series of mono- and di-substituted piperazine derivatives have been synthesized. For di-substituted derivatives, ciprofloxacin was selected and hybrids were synthesized via substitution at piperazinyl-N4. In vitro antibacterial studies of all synthesized compound were carried out against American Type Culture Collection (ATCC) strains; E. coli (ATCC 25922), P. aeruginosa (ATCC 15442), K. pneumoniae (ATCC 1705), B. subtilis (ATCC 6633) and S. aureus (ATCC 6538). The potent series of compounds were further evaluated for their potential against clinically isolated resistant strains of E. coli, P. aeruginosa, S. aureus, and S. hemolytic. The reaction of piperazinyl-NH of ciprofloxacin with selected drugs resulted in pronounced growth inhibition of standard as well as resistant bacterial strains. Hybrid compounds 14b, 16a, 16d and CGS-20 showed excellent bacterial growth inhibition against standard and resistant strains. In vitro results were further correlated by using in silico tools. Molecular docking studies were carried out using MOE (Molecular Operating Environment) software. DNA gyrase used as a target and all compounds were docked against this specific target.