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The folate biosynthetic pathway offers many druggable targets that have yet to be exploited in tuberculosis therapy. Herein, we have identified a series of small molecules that interrupt Mycobacterium tuberculosis (Mtb) folate metabolism by dual targeting of dihydrofolate reductase (DHFR), a key enzyme in the folate pathway, and its functional analog, Rv2671. We have also compared the antifolate activity of these compounds with that of para-aminosalicylic acid (PAS). We found that the bioactive metabolite of PAS, in addition to previously reported activity against DHFR, inhibits flavin-dependent thymidylate synthase in Mtb, suggesting a multi-targeted mechanism of action for this drug. Finally, we have shown that antifolate treatment in Mtb decreases the production of mycolic acids, most likely due to perturbation of the activated methyl cycle. We conclude that multi-targeting of the folate pathway in Mtb is associated with highly potent anti-mycobacterial activity.