The reaction of non-symmetrically substituted N-benzyl benzimidazolium salts (1–3) with Ag2O gives Ag(I)–NHC complexes (4–6) of the type [NHC–Ag–NHC]PF6, (NHC: benzimidazol-2-ylidene). Structures of these compounds were elucidated by physico-chemical and spectroscopic techniques. The mononuclear structures of complexes 4 and 5 in the solid state were further confirmed by single-crystal X-ray diffraction studies. The effect of substitutions on antibacterial activities of compounds 1–6 by successive N-alkylation has been investigated. The compounds were screened for their antibacterial efficacy against Staphylococcus aureus (ATCC 12600) as a Gram-positive bacterium and Escherichia coli (ATCC 25922) as a Gram-negative bacterium. All the Ag(I)–NHC complexes, in general, showed good antibacterial activities while their corresponding salts were inactive against both strains of bacteria. The results of this investigation showed that E. coli and S. aureus appeared markedly inhibited. It was observed that the derivatives of the Ag(I)–NHC complexes with longer alkyl chains were more bioactive. In order to gain preliminary insights into their actual mode of action(s), circular plasmid pTS414 DNA was exposed to gel electrophoresis and it was found that all the complexes were efficient in promoting the cleavage or degradation of DNA in the absence of an oxidant.