Transcription in prokaryotes is a multistep process and is primarily regulated at the initiation stage. σ factors are involved in promoter recognition and thus govern prokaryotic gene expression. Mycobacterium tuberculosis (Mtb) σ factors have been previously suggested as important drug targets through large-scale genome analyses. Here we demonstrate the feasibility of specific targeting of Mtb σ factors using designed peptides. A peptide library was generated using three-dimensional structural features corresponding to the interface regions of σ factors and the RNA polymerase. In silico optimization of the peptides, employing structural as well as sequence features, aided specific targeting of σA and σB. We synthesized and characterized the best hit peptide from the peptide library along with other control peptides and studied the interaction of these peptides with σB using biolayer interferometry. The experimental data validate the design strategy. These studies suggest the feasibility of designing specific peptides via in silico methods that bind σB with nanomolar affinity. We note that this strategy can be broadly applied to modulate prokaryotic transcription by designed peptides, thereby providing a tool for studying bacterial adaptation as well as host-pathogen interactions in infectious bacteria.