IMPORTANCE: A previous study on the influence of usnic acid on the avirulent H37Ra strain revealed that the early bacterial response was associated with redox homeostasis, lipid synthesis, and nucleic acid repair. The response of bacteria to antimicrobials is specific to each species and strain. Given the genetic and phenotypic differences between the avirulent H37Ra strain and the virulent H37Rv strain, we combined lipidomics and global transcriptomics to uncover the mechanism of action of usnic acid against H37Rv. The study identified strain-specific differences between the virulent H37Rv and avirulent H37Ra. The H37Ra strain exhibited increased metabolic activity, while the H37Rv strain showed a reduction in basic metabolic processes and activated alternative iron-dependent energy production. These differences highlight the varying susceptibility of sister strains within the same species to the same antibacterial agent.