The DNA replication machinery of the Plasmodium falciparum apicoplast is a validated drug target. Nuclear-encoded gyrase subunits are predicted to play a critical role in maintaining DNA topology during the D-loop/bi-directional ori replication process of the parasite. We show the presence of P. falciparum gyrase subunits in parasite lysates by using antibodies generated against recombinant gyrase A and B. The ATPase activity of PfGyrB was inhibited by novobiocin that also caused parasite death in culture. Reduction of apicoplast/nuclear DNA ratio in the presence of novobiocin indicated that the drug targets apicoplast DNA replication. Molecular modeling of gyrase A and B subunits revealed extensive fold conservation with the Escherichia coli counterparts as well as the presence of a long disordered loop adjacent to the ATPase domain of PfGyrB. Our results have implications for development of PfGyrB as a drug target against malaria.