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Nosocomial and community-acquired infections due to pseudo and Gram-positive bacteria such as Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus (MRSA) respectively, are associated with high levels of mortality, morbidity and significant social and economic costs in the UK and elsewhere. As the evolution of multi-drug resistance relentlessly erodes the utility of currently available antibacterial drugs, it is essential to maintain efforts to search for new classes of antibacterial agents. Benzimidazoles are potent anti-helmintic agents discovered in 1961. Chemical modifications led to the synthesis of the head-to-tail fluorochrome Hoechst 33258 and, more recently, to symmetric head-to-head bis-benzimidazoles (sBBZs). The antibacterial potential of these compounds was examined. Antibacterial activity was evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays using a total of 143 Gram-positive isolates, including 61 MRSA, 12 vancomycin resistant enterococci (VRE), 12 Streptococcus pneumoniae, 11 Listeria monocytogenes, 7 Mycobacterium spp. (including Mycobacterium tuberculosis) and 14 Gram-negative isolates. Structure-activity relationships were determined and key requirements for activity were identified. Acute toxicity of sBBZs was assessed using the sulforhodamine B staining method and a zebrafish embryo model. To clarify mode of action, time kill studies, flow cytometry, DNA microarrays and radioisotope incorporation assays were employed. sBBZs displayed activity against Gram-positive but not Gram-negative pathogens; the most potent compound possessed MIC90 values of 0.06, 0.125 and 1 mg/L against, respectively, MRSA, VRE and M. tuberculosis isolates. Data suggests that sBBZs are bacteriostatic agents that interfere primarily with the DNA machinery and do not select for drug resistant variants over a 35-day period of drug exposure. Subtle structural modifications have an incisive effect on in vitro potency of sBBZs. Toxicity was determined as minimal for WI-38 cell line and the zebrafish embryo model of infection. Sub-inhibitory concentrations of sBBZs inhibited MRSA transcription of pathogenicity-associated genes. Thus, symmetric 6/5-benzimidazoles are new DNA-interfering bacteriostatic agents with potent antibacterial activity and significant therapeutic potential against Gram-positive bacteria, including MRSA and M. tuberculosis.