The synthesis of silver nanoparticles (AgNPs), using plant extracts, bacteria, fungi and yeasts, and their antimicrobial activities have been widely investigated and well documented. However, pullulan AgNPs and their antimicrobial activities have not received much attention. The objective of this study was to synthesize pullulan AgNPs, characterize them, and test their antibacterial and antifungal activities. Pullulan was extracted from Aureobasidium mangrovei isolated from Oman and, using UV-Vis spectroscopy and Fourier Transform Infrared (FT-IR), found to be identical to the commercial pullulan obtained from Sigma, USA. Transmission electron microscopy (TEM) showed that most of the synthesized particles were poly-dispersed, irregular in shape, and most were spherical with an average size of 9.76 nm. Pullulan-mediated AgNPs were found to have antibacterial activities, and the ANOVA test showed that there were no significant differences between AgNO3, Pullulan and pullulan-mediated AgNPs for all the bacteria tested. Pullulan-mediated nanoparticles were found to have antifungal activity against Curvularia lunata, Fusarium incarnatum, Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus and Penicillium sp. The ANOVA test also revealed that there was a significant difference in antifungal activity between pullulan and pullulan-mediated AgNPs, pullulan-mediated nanoparticles having shown a higher inhibitory activity than pullulan. Pullulan and pullulan-mediated nanoparticles could be used in the food industry and are safer than silver nitrates.