Twelve marine-derived fungal strains were evaluated for their ability to synthesize silver nanoparticles (AgNPs). Mycogenic AgNPs were preliminarily characterized using different techniques, and their antimicrobial activities were assessed. Penicillium citrinum IBCLP11 and Aspergillus niger IBCLP20 were selected for AgNPs’ synthesis optimization by varying parameters such as AgNO₃ concentration, biomass, agitation, temperature, and pH. AgNP_IBCLP11 and AgNP_IBCLP20 were able to inhibit the growth of Pseudomonas aeruginosa IPT322, Staphylococcus aureus IPT246, and Klebsiella pneumoniae IPT412 at concentrations of 25 μg/mL or higher. Aspergillus niger IPT295 and Aspergillus parasiticus IPT729 were the most sensitive to AgNP_IBCLP20. Further studies are needed to fully elucidate the effects of all parameters influencing mycogenic AgNPs synthesis. However, it is evident that maintaining optimal conditions, such as temperature and pH during agitation, is crucial for preventing undesirable reactions and ensuring nanoparticle stability.