The structure activity relationships of 1H-benzimidazole-8-carboxylic acids (9a-9f), 1H-benzimidazole-8-carboxylates (8a-8f) are described. Characteristic compounds showed improved demanding activity over a previously recognized 2-aminobenzimidazole series. In the lead optimization process and structure–activity relationship (SAR) of separate inhibitors based on modification of the lead molecule (E)-1,6-dimethyl-9-oxo-2-styryl-6,9-dihydro-1H-imidazo[4,5-f]quinoline-8-carboxylic acids, identified from a high-throughput-screen. Noticeably, 4-chlorobenzyl, 4-fluorobenzyl benzimidazole derivatives 9c, 9e gave remarkable antimicrobial activities against saccharomyces cerevisiae, MRSA and bacillus proteus with MIC values of 1, 2 and 4 μg/mL, respectively. Experimental research revealed that compound 5c could effectively intercalate into calf thymus DNA to form compound 5c DNA complex which might block DNA replication and thus exert antimicrobial activities.