Silver particles were prepared by dewetting Ag films coated on glass using a fiber laser. The size of the particles was controlled in the range of 92 nm–1.2 μm by adjusting the thickness of the Ag film. The structural properties and surface roughness of the particles were evaluated by means of scanning electron microscopy. In addition, the antifungal activity of the Ag particles was examined using spore suspensions of Colletotrichum gloeosporioides. It is shown that particles with a size of 1.2 μm achieved 100% inhibition of conidia growth of C. gloeosporioides after a contact time of just 5 min. Furthermore, the smaller particles also achieved good antifungal activity given a longer contact time. Similar results were observed for spore germination and pathogenicity tests performed on mango fruit and leaves. Overall, the results confirm that Ag particles have an excellent antifungal effect on C. gloeosporioides.