This study explores the potential of silver nanoparticles (AgNPs) synthesized through an eco-friendly method using coffee extract to combat non-bacterial mastitis in dairy cattle. Mastitis, often caused by pathogens such as yeasts and algae like spp., poses a challenge due to the limited efficacy of traditional antibiotics. This research utilized strains isolated from mastitis milk and assessed the nanoparticles' physicochemical properties, antimicrobial efficacy, and impact on biofilm formation and microorganism invasion. AgNPs demonstrated a spherical shape with a mean hydrodynamic diameter of ~87 nm and moderate colloidal stability. Antimicrobial tests revealed significant growth inhibition of yeast and spp., with minimal inhibitory concentrations (MICs) as low as 10 mg/L for certain strains. Biofilm formation was notably disrupted, and microorganism invasion in bioprinted gels was significantly reduced, indicating the broad-spectrum potential of AgNPs. The study highlights the nanoparticles' ability to damage cell membranes and inhibit metabolic activities, presenting a promising alternative for managing infections resistant to conventional treatments. These findings suggest that green-synthesized AgNPs could play a pivotal role in developing sustainable solutions for mastitis treatment, particularly for pathogens with limited treatment options.