A novel highly substituted and fluorescent aromatic-fused imidazole derivative has been synthesized by rational design. This novel fluorescent material acts as an alternative antibacterial agent against Gram positive bacteria strains. It shows superior antibacterial activity (with MIC value of 8 mu g/mL) against methicillin-resistant Staphylococcus aureus (MRSA) when compared with standard antibiotic drugs Ampicillin (with MIC value of 128 mu g/mL) and Kanamycin (with MIC value of > 512 mu g/mL). The interaction of this novel compound with the bacterial cell and genomic DNA has also been studied to elucidate antibacterial mode of action. Fluorescence spectroscopy and microscopy studies have proved the intracellular uptake of this special compound. Likewise, UV-vis and fluorescence spectroscopy studies have revealed a significant decrease in the absorption and emission bands of the compound upon its interaction with plasmid and genomic DNA, which is likely due to its DNA intercalation property. Furthermore, these findings have been supported by gel electrophoresis of genomic DNA of S. aureus cells treated with the compound. The results indicate that this novel compound exerts its antibacterial activity by causing DNA damage, suggesting the potential utility of fluorescent probes for real-time diagnosis and treatment of bacterial infections.