Bacterial infections create distinctive microenvironments with a unique mix of metabolites and enzymes compared with healthy tissues that can be used to trigger the activation of antibiotic prodrugs. Here, a single and dual prodrug masking the C3 carboxylate and C7 piperazine of the fluoroquinolone, ciprofloxacin, responsive to nitroreductase (NTR) and/or hydrogen sulfide (H2S), was developed. Masking both functional groups reduced the activity of the prodrug against Staphylococcus aureus and Escherichia coli, increasing its minimum inhibitory concentration (MIC) by ∼512-fold (S. aureus) and ∼8000-fold (E. coli strains), while masking a single group only increased the MIC by ∼128-fold. Bacteria subjected to prolonged prodrug exposure did not show any increase in resistance. Triggering assays demonstrated the conversion of prodrugs to ciprofloxacin, and in a murine infection model, responsive prodrugs showed antibacterial activity comparable to that of ciprofloxacin, suggesting in vivo activation of prodrugs. Thus, the potential for site-specific antibiotic treatment with reduced threat of resistance is demonstrated.