Substrate-based sirtuin inhibitors target bacterial genome and RNA and provide a promising approach to address bacterial resistance issues, if cellular internalisation can be achieved. We designed N-trifluoroacetyl lysine and N-thioacetyl lysine peptides (KP 13, KP 15 and KP 24) as inhibitors of bacterial sirtuins and their cell-penetrating peptide conjugates Tat KP 13, Tat KP 15 and Tat KP 24. The conjugated peptides were successfully internalised and showed signs of bacterial transcription inhibition resulting in enhanced antibacterial potency against model Gram negative and Gram positive pathogens. Synergistic activity in combination with streptomycin and polymyxin B has also been established. These peptides were effective in inhibiting biofilm formation and eradicating preformed biofilms. Morphological analysis using both SEM and TEM showed bacterial membrane disruption. Calcein dye leakage analysis established the selectivity of these peptides to bacterial membranes. This study documents the first report of the application of substrate-based sirtuin inhibitors as antimicrobial therapeutics.