Herein we report the green synthesis of silver nanoparticles (EcAgNPs) from water fraction (Ecs), obtained from methanol extract of Eucalyptus camaldulensis at room conditions. UV-vis, HR-TEM and SEM-EDS devices were used to characterize the EcAgNPs. The UV-visible spectrum of EcAgNPs showed surface plasmon resonance peak at 420 nm. HR-TEM images showed that the EcAgNPs were spherical with a diameter in the range 3.7-29.6 nm. Ag+ ions were reduced to Ago by phytochemicals in the without adding external reducing agent. The presence of biomolecules in the Ecs was investigated by UPLC-MS/MS that detected gallic acid (20.26±0.10 ppb) and quercetin (12.4±0.08 ppb) as major constituents. The antimicrobial activities of Ecs and synthesized EcAgNPs were tested on Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus strains and Candida albicans yeast. Ecs exhibited antimicrobial activity as MIC between 16.2 and 129.3 mg mL-1 while EcAgNP showed MIC between 6.31 and 14.65 µgmL-1. The cytotoxicity of both Ecs and EcAgNPs on A549, HT29 and MDA-MB-231 cancer cell lines was tested by MTT. EcAgNPs showed more significant anticancer activity on MDA-MB-231, HT29 and A549 cancer cell lines (8.10±0.01, 5.08±0.01 and 18.58±0.03 µg mL-1) than Ecs (219.70±0.73, 916.24±0.67, 999.30±1.86 µg mL-1) and it should be investigated for use in various therapeutics.