Silver nanoparticles contribute a giant share to the realm of modern nanobiotechnology. Their utility as antimicrobial agents is also well documented. Green synthesis of nanoparticle has several advantages over its chemical synthesis. In the present study, Thuja occidentalis leaf extract mediated silver nanoparticles were prepared without using a stabilizing agent and tested for their anticancer and anti-microbial activity. Thuja occidentalis leaf extract mediated silver nanoparticles were prepared under ambient conditions which showed a narrow size distribution within the range of 10–15 nm, with average particle size of 12.7 nm. Interestingly, these nanoparticles exhibited anti-cancer activity against human breast (MCF 7, MDA MB 231) and cervical cancer (HeLa) as well as mouth epidermoid carcinoma (KB) cell lines at a concentration range of 6.25–50 μg/mL. Contrarily, they are compatible with human peripheral blood mononuclear cells and rat hepatocytes. Moreover, their efficient inhibitory effect was witnessed against Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa with inhibitory concentration at 5–10 μg/mL. The prepared nanoparticles were highly biocompatible and have strong potential in the development of non-toxic chemotherapy with antibacterial attributes.