Body of Abstract: Currently, drug resistance has emerged as a major problem for various infections. In such cases, plants can be used as an alternative to producing new antimicrobial agents. This study examined the antibacterial activity of Thuja arborvitae extract on gram-positive (Staphylococcus aureus and Streptococcus spp.) and gram-negative bacteria (E. coli and Pseudomonas aeruginosa). In Nepal, people have treated bacterial infection by the extraction of Thuja leaves. We discovered the actual chemical structure of this plant exhibiting bioactivity against these different microorganisms. The antibacterial activity was tested by the zone of inhibition (ZOB) and minimum inhibition concentration (MIC) methods. Thuja-extracted compounds were purified through conventional and HPLC chromatography methods. We found that the Thuja leaf contains four distinct antibiotic compounds, which were named “P1,” “P2,” “P3,” and “P4.” These four antibiotic compounds all showed potent antibacterial activity against the tested microorganisms. The chemical structures of P2, P3, and P4 compounds have been solved and further purification of P1 is being done. We propose that the Thuja leaves have antibacterial effects and can be a potential source for producing antibacterial drugs. Based on these results this study promises an interesting future for designing a potential antibiotic that could replace resistant antibiotics used today.