Abstract:[Objective] To biosynthesis silver nanoparticles using Trichoderma hamatum and detect antibacterial activity of silver nanoparticles. [Methods] Silver nanoparticles were biosynthesized using Trichoderma hamatum mixed with AgNO3 and were characterized by UV-vis, XRD and TEM. Thermogravimetric analysis and atomic absorption were used to detected productivity of silver nanoparticle and conversion rate of Ag+. Antibacterial activity of silver nanoparticles was detected using Escherichia coli and Bacillus subtilis. [Results] Culture solution of Trichoderma hamatum mixed with AgNO3 was reddish-brown; UV-vis spectrum showed a significant peak at 420 nm; XRD spectrum showed four peaks, which corresponded to four crystal faces of silver nanoparticles; TEM images suggested that silver nanoparticles were monodisperse with shape in spherical. Size distribution suggested that they had narrow distribution, between 1–13 nm and the average size was 6.69 nm. Atomic absorption showed conversion rate of Ag+ was 84.41%. Thermogravimetric analysis showed that the productivity of silver nanoparticles was 67.12%. MBC of silver nanoparticle for E. coli was 10 mg/L and MIC was 7 mg/L, while for Bacillus subtilis was 5 mg/L and 4 mg/L. [Conclusion] After mixed with AgNO3, Trichoderma hamatum can biosynthesis silver nanoparticles. Silver nanoparticles were uniform, cubic and pure. Lethal effect of silver nanoparticles for Bacillus subtilis was much greater than that for E. coli.