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Natural polysaccharides are attractive and promising biomacromolecules for the green synthesis of silver nanoparticles (Ag NPs) with a broad spectrum of useful functions. This study aims to evaluate the synthetic conditions and physical properties of Ag NPs using three fractions of exopolysaccharide (EPS), namely EPS-1, EPS-2, and EPS-3, produced by a medicinal fungus known as Cs-HK1, with variations in their chemical composition and molecular weight. Each of the EPS fractions had a unique set of optimal synthetic conditions (reaction time course, temperature, and reagent concentration), resulting in a specific range of Ag NP size distributions. The Ag NPs synthesized with the EPS-1 fraction had the smallest particle size (~160 nm) and the most significant antibacterial activities against <i>Escherichia coli</i> (Gram−) and <i>Staphylococcus aureus</i> (Gram+), with a minimal inhibitory concentration (MIC) of 0.2 mg/mL on <i>E. coli</i> and 0.075 mg/mL on <i>S. aureus</i>. The results proved the success of the scheme of this green synthesis scheme with all three EPS fractions and the potential antibacterial application of EPS-coated Ag NPs.