Tuberculosis has become a cause of worldwide concern; emergence of resistance in various mycobacterial strains has led to an urgent demand for new therapeutic molecules. Antimicrobial peptides have emerged as potential candidates in antimicrobial drug development. We have tested a lead antibacterial peptide LP-23 and its cost-effective mimetic DP-23 for their antimicrobial effects in a non-pathogenic model of Mycobacterium. Their minimum inhibitory concentration (MIC) values against M. smegmatis were calculated by resazurin reduction assay. MIC of both peptidomimetics against M. smegmatis was found to be 6.25 μg/mL. In addition, hemolytic toxicity study of peptidomimetics suggested that the synthesized compounds were selective against bacteria. To better understand stability and mechanism of action of peptidomimetics, serum stability study and SEM analysis were carried out. Peptoid DP-23 was found to be more stable in serum then peptide LP-23, while SEM analysis indicated that these compounds target the cell membrane, impairing the membrane integrity of M. smegmatis. The activity and properties of LP-23 and DP-23 may make them new potential antibacterial agents against tuberculosis.