In bacteria, Rho-dependent transcription termination plays a major role in the regulation of gene expression. It regulates many physiological processes directly or indirectly making the conserved Rho protein a very attractive drug target. The Psu protein is a capsid protein in bacteriophage P4 that moonlights as a specific inhibitor of Rho. Expression of Psu induces lethality to Escherichia coli as well as many other pathogens. Here, we report the generation of peptide inhibitors against Rho from the Psu-CTD through a directed evolution method. We screened peptides capable of inhibiting in vivo Rho-dependent termination as well as inducing lethality in a similar way as Psu. In vitro, the peptides inhibited the RNA binding, RNA release and ATPase activity by Rho. Direct interaction of the peptides with Rho was evident from pull down and ITC assays. Deletion of 8 amino acids from the C-terminal of the peptide did not affect its function. The N-terminal Histidine tag was important for their solubility in vitro and presumably in vivo. The ITC assays indicated that the peptides occupy the N-terminal domain of four of the subunits of Rho. Thus, the mode of interaction of the peptides is different from that of Psu. These peptides are also lethal against the Mycobacterium smegmatis and Mycobacterium bovis. In vitro, the peptides could inhibit the ATPase activities of Mycobacterium tuberculosis and the plant pathogen, Xanthomonas orizae Rho proteins. Our results establish an alternative way to design new bactericidal molecules using the bacteriophage proteins as platforms.