Tuberculosis remains one of the top causes of death worldwide and combatting its spread has been severely complicated by the emergence of drug-resistance mutations highlighting the need for more effective drugs. Despite the resistance to isoniazid (INH) arising from mutations in the katG gene encoding the catalase-peroxidase KatG, most notably the S315T mutation, this compound is still one of the most powerful first-line antitubercular drugs, suggesting further pursuit of the development of tailored INH derivatives. The N'-acylated INH derivative with a long alkyl chain (INH-C10) has been shown to be more effective than INH against the S315T variant of Mycobacterium tuberculosis, but the molecular details of this activity enhancement are still unknown. In this work, we show that INH N'-acylation significantly reduces the rate of production of both isonicotinoyl radical and isonicotinyl-NAD by wild type KatG, but not by the S315T variant of KatG mirroring the in vivo effectiveness of the compound. Restrained and unrestrained MD simulations of INH and its derivatives at the water/membrane interface were performed and showed a higher preference of INH-C10 for the lipidic phase combined with a signicantly higher membrane permeability rate (27.9 cm s(-1)), compared with INH-C2 or INH (1.3 and 3.8 cm s(-1), respectively). Thus, we propose that INH-C10 is able to exhibit better minimum inhibitory concentrations (MIC) values against certain variants because of its greater affinity for the lipid membrane which enhances its passage into the cell. MIC values of INH and INH-C10 against two additional KatG mutations (S315N and D735A) revealed that some KatG variants are able to process INH faster than INH-C10 into an effective antitubercular form (wt and S315N), while others show similar reaction rates (S315T and D735A). Altogether, our results highlight the potential of increased INH lipophilicity for treating INH-resistant strains.