Silver nanoparticles (AgNPs) were biosynthesized using Fortunella Japonica extract as an reducing agent through a rapidly and ecofriendly assisted ultrasound method. Effect of ultrasound assistant on synthesis duration, properties and antibacterial activity of AgNPs against Escherichia coli, Bacillus subtilis and Bacillus cereus were reported. The suitable duration for AgNPs assisted ultrasound biosynthesis was 90 minutes that is 60 minutes shorter than stirring-assisted method. The obtained AgNPs with a face centred cubic structure were nearly spherical in shape and uniform in size distribution with the average nanosize of 11.6 nm, being smaller than the size of AgNPs prepared by stirring-assisted method. AgNPs sample exhibited effective antibacterial activity against all three bacteria with average diameter of inhibition zones of over 15 mm and minimum inhibitory concentration of 4.13 µg/mL. The results showed that the ultrasound assistant has positive effects such as shortening synthesis duration, reducing average particle size and increasing antibacterial activity of AgNPs.