Identification of compounds distinguishing between NAD⁺ and ATP-dependent ligases is a key factor to discover new antimicrobials. Based on virtual screening experiments a series of hydroxamates distinguishing between the above two classes of enzymes are synthesized. The specificity of the compounds for the bacterial LigA was tested in vivo involving a temperature sensitive LigA deficient E. coli GR501 strain rescued with the M. tuberculosis LigA (MtuLigA) and a wild type S. typhimurium and its LigA-strain rescued with T4 ligase. The compounds exhibit similar specificity for the NAD⁺-dependent ligases. In vitro assays involving the MtuLigA and the human DNA ligase I enzymes show that two compounds are ∼8-fold specific at low μM concentrations. Ethidium bromide displacement assays indicate that the compounds do not exhibit general interactions with DNA. The overall study suggests that hydroxamates have the potential to be developed as novel antibacterials.