- Mycobacterium Tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of tuberculosis (TB) that kills more than 1.5 million people worldwide annually. One of the main reasons for this high mortality rate is the evolution of new Mtb strains that are resistant to available antibiotics. Therefore, new therapeutics for TB are in constant demand. It is known that DNA replication enzymes of Mtb and humans are different and this represents a potential target for anti-Mtb drug development. Here we report the development of potent inhibitors that target simultaneously two DNA replication enzymes of Mtb, namely DnaG primase and DNA gyrase, which share a conserved fold near the inhibitors′ binding site. In order to improve the physicochemical properties, the binding affinity and inhibitory activity against these two enzymes, 49 novel compounds were synthesized as potential drug candidates in three stages. The last stage of chemical optimization yielded two novel selective inhibitors that exhibit bacteriostatic activity against Mycobacterium smegmatis (Msmg), a fast growing non-pathogenic model specie of Mycobacteria. Efficacy was improved upon conjugation of the best candidate to a cell penetrating peptide (CPP). The inhibitors also impair the development of biofilm, indicating a therapeutic potential of these molecules against infections caused by planktonic and sessile forms of mycobacterium species.