Emergence of antibiotic resistance by bacteria has become a serious threat for public health worldwide. In this study, Streptomyces isolated from fertile soil sample was tested for biosynthesis of silver nanoparticles (AgNps) using cell-free supernatant and synthesized AgNps were screened for anti-ESBL (extended spectrum b-lactamase) activity against multi-drug resistant (MDR) ESBL-producing strain Klebsiella pneumoniae (ATCC 700603) and other medically important pathogens. Synthesis of AgNps was confirmed by change in pale yellow color to dark brown color and characteristic absorption spectra at 420 nm. The XRD spectrum displayed typical peaks of crystalline silver and EDAX analysis showed a major signal for silver. FTIR spectra revealed prominent peaks at 3,294 cm-1 (NH stretching due to amide group), 2,952 cm-1 (aldehydic C-H stretching) 1,658 cm-1 indicating the presence of carbonyl group. AgNps were spherical in shape with size ranging from 20 to 70 nm. The synthesized AgNps showed significant antimicrobial activity against standard ESBL pathogen K. pneumoniae (22 mm), 21 mm against clinical ESBL isolate E. coli and 16 mm against clinical ESBL isolates K. pneumoniae and Citrobacter species, respectively. The results of this study suggest that AgNps synthesized by Streptomyces sp. VITSJK10 can be used as a potential alternative to control MDR ESBL pathogens. The present study aimed for green synthesis of AgNps using Streptomyces species and to explore its anti-ESBL activity. © Springer-Verlag 2013.