On the basis of recently reported 1-hydroxybenzoyl-4-arylthiosemicarbazides and 4-benzoyl-1-dichlorobenzoylthiosemicarbazides, three series of chemically modified 1,4-dibezoylthiosemicarbazide derivatives were synthesized and their antibacterial activity was tested. The most potent of studied compounds had MICs≤8μg/mL against reference strains of Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, and Micrococcus luteus. Further studies showed that selected compounds inhibited the growth of most Staphylococcus aureus clinical isolates at concentrations equipotent or even lower than that required for ampicillin and proved in some cases to be as effective or even more than vancomycin, streptomycin, and nitrofurantoin. From a comparison of the results of toxicity and antibacterial activity it is evident that the most potent antibacterial agents are nontoxic toward mammalian cells at their MIC values.