6-Anilinopyrazolo[3,4-d]pyrimidin-4-ones are novel dGTP analogues that inhibit the replication-specific enzyme DNA polymerase III (DNA pol III) of Staphlococcus aureus and other Gram-positive (Gr+) bacteria. To enhance the potential of these inhibitors as antimicrobial agents, a structure-activity relationship was developed involving substitutions at the 2, 4, and pyrazolo NH positions. All of the new inhibitors were tested for their ability to inhibit S. aureus DNA pol III and the growth of several other Gr+ bacteria in culture. 2-Anilino groups with small hydrophobic groups in the meta or para position enhanced both antipolymerase and antimicrobial activity. 2-Benzyl-substituted inhibitors were substantially less active. Substitution in the 4-position by oxygen gave the optimal activity, whereas substitution at the pyrazolo NH was not tolerated. These pyrazolo[3,4-d]pyrimidine derivatives represent a novel class of antimicrobials with promising activities against Gr+ bacteria.