The ability of Uropathogenic Escherichia coli (UPEC) to form biofilms, can be considered an important factor that contributes to the prevalence of Urinary Tract Infections (UTIs) due to the inaccessibility of the antibiotics into the highly complex structure of the biofilm. Moreover, with the appearance of antibiotic multiresistant UPEC strains, the alternatives of treatment of UTIs are less. Silver nanoparticles (AgNPs) can be useful in the treatment of the UPEC infections due to its physicochemical properties that confer them antibacterial activity against both planktonic and biofilm structured cells. A diversity of biological methods for synthesis of AgNPs with antimicrobial activity has been widely investigated during the last decades, between these methods; the fungal-biosynthesis of AgNPs highlights as an ecofriendly, scalable and low cost method. In this study, biogenic AgNPs were synthesized with extracellular metabolites secreted by the soil fungal strain Fusarium scirpi (Ag0.5-5) by an ecofriendly, simple and efficient method. The antimicrobial activity of the biosynthesized AgNPs against UPEC was evaluated. The Minimal Inhibitory Concentration (MIC) of biogenic AgNPs over planktonic UPEC cells was 25 mg/mL, whereas a sub-MIC concentration (7.5 mg/L) was sufficient to inhibit the UPEC-biofilm formation about a 97%, or produce the disruption of an 80% of mature UPEC-biofilms demonstrating the potential of fungal-derived AgNPs to prevent UPEC infections.