Antibiotic resistance is one of the most important global problems currently confronting the world. Different biomedical applications of silver nanoparticles (AgNPs) have indicated them to be promising antimicrobial agents. In the present study, extracellular extract of an endophytic bacterium, Pantoea ananatis, was used for synthesis of AgNPs. The synthesized AgNPs were characterized by UV–Vis spectroscopy, FTIR, transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), and Zeta potential. The antimicrobial potential of the AgNPs against pathogenic Staphylococcus aureus subsp. aureus (ATCC 11632), Bacillus cereus (ATCC 10876), Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 10145) and Candida albicans (ATCC 10231), and multidrug resistant (MDR) Streptococcus pneumoniae (ATCC 700677), Enterococcus faecium (ATCC 700221) Staphylococcus aureus (ATCC 33592) Escherichia coli (NCTC 13351) was investigated. The synthesized spherical-shaped AgNPs with a size range of 8.06 nm to 91.32 nm exhibited significant antimicrobial activity at 6 μg/disc concentration against Bacillus cereus (ATCC 10876) and Candida albicans (ATCC 10231) which were found to be resistant to conventional antibiotics. The synthesized AgNPs showed promising antibacterial efficiency at 10 µg/disc concentration against the MDR strains. The present study suggests that AgNPs synthesized by using the endophytic bacterium P. ananatis are promising antimicrobial agent.