The emergence and spread of multidrug-resistant strains of Staphylococcus aureus and Mycobacterium tuberculosis are generating a threat to public health worldwide. In the current study, a series of N(1)-benzyl and N(1)-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamine derivatives were synthesized and investigated for their antimicrobial activity against S. aureus, and Mycobacterium smegmatis which is taxonomically related to M. tuberculosis. Most of the compounds exhibited good activity against M. smegmatis as determined by comparison of diameters of the zone of inhibition of test compounds and standard antibiotics. Compound 7o showed potent antimycobacterial activity against M. smegmatis without mammalian DHFR inhibition liability. The results from this study indicate that 1-benzyl derivatives of 1,6-dihydro-1,3,5-triazine-2,4-diamines may be used as lead compounds for the discovery of antimycobacterial agents.