The methanolic extracts from fruit, leaf, stem and roots of Paeonia daurica subsp. macrophylla (P. daurica) were investigated for inhibitory effect on replicative bacterial (PolC and DnaE2) and viral (MMLV-RT from Moloney Murine Leukemia Virus) DNA polymerases by primer extension assay. While all plant parts showed inhibition effect on bacterial and viral DNA polymerases, roots of the plant was focused to purify inhibitory compound(s). The chemical structures of compounds were completely elucidated using a combination of NMR, MS and FT-IR analyses. Five molecules with tree monoterpene glycosides, paeoniflorin (PD-2), paeoniflorigenone (PD-4), benzoyl paeoniflorin (PD-5), and benzoic acid (PD-3) with its derivate 2,4,6-trihydroxy-1-methyl benzoate (PD-1) were purified and identified. Both DNAdependent and RNA-dependent polymerase activity of MMLV-RT was strongly inhibited by these five molecules. On the other hand, bacterial polymerases PolC and DnaE2 were strongly inhibited by only paeoniflorigenone (PD-4). Molecular modeling result suggested that paeoniflorigenone (PD-4) interacts with the important residues at active site (palm, fingers and thumb domains) of three polymerases which support our experimental result. Ethyl acetate fraction had smallest SC50 value against DPPH and ABTS radicals. It showed also higher scavenging activity than quercetin, trolox and ascorbic acid since its quite high total phenolic content. We proposed that the parts of P. daurica might be used to find new antimicrobial agents and generate supplementary material for foods. Furthermore, the isolated molecules with inhibitory effect may be used as new scaffold for the further modification in order to develop novel inhibitors against DNA polymerization.