Thirty-three new quaternization harman analogues were synthesized and their antibacterial activity against four Gram-positive and two Gram-negative bacteria were evaluated. The structure-activity relationships were summarized and compounds 4f, 4i, 4l, 4u, 4w, 4x and 5c showed excellent antibacterial activity, low cytotoxicity, good thermal stability and "drug-like" properties. In particular, compound 4x exhibited better bactericidal effect (4-fold superiority against methicillin-resistant Staphylococcus aureus) than standard drugs fosfomycin sodium and ampicillin sodium (minimum inhibitory concentration = 50 nmol/mL). Scanning electron microscopy revealed morphological changes of the bacterial cell surface and the docking evaluation provided a good total score (6.4952) for 4x which is close to the score of ciprofloxacin (6.9723). The results indicated that the quaternization harman analogues might exert their bactericidal effect by damaging bacterial cell membrane and wall, and disrupting the function of type II topoisomerase. In addition, the in vivo antibacterial assay with a protective efficacy of 81.3% further demonstrated the potential of these derivatives as new bactericides and antibiotics.