Antibacterial activity of fifteen piperazine-1-carbodithioate derivatives of 2,4-diaminoquinazoline was investigated in order to search for compounds with efficient antibacterial activity. First, the binding energies of substrate-enzyme were predicted by molecular docking calculation. Second, minimum inhibitory concentration (MIC) values of the tested compounds were measured against the multidrug-resistant Escherichia coli (E. coli) with a standard method. The results showed that some compounds had better antibacterial activity than the control, and MIC of compounds 7a, 7g and 7k was below 1 mg/mL. The results were further confirmed by high performance capillary electrophoresis (HPCE) screening method, and the half inhibitory concentration (IC₅₀) of 7k was 4.97×10^－2 mg/mL. In addition, structure-activity relationship analysis showed that the presence of an electron-withdrawing group at the C2-position of phenyl ring linked with piperazine moiety was favorable to the antibacterial activity. This study provided a new option for the development of antimicrobial agents.