Mycological synthesized silver nanoparticles (SNPs) is a promising field of study and addressing the optimized conditions for this production is discussed in detail in this study. Here, we screened 80 novel fungal isolates, obtained from Padina sp. (Algicolous fungi) and Avicennia marina (Forssk.) Vierh. (Foliicolous fungi) samples, for SNPs generation using fungal supernatants to identify new isolates with enhanced activity. A simple new small-scale method for screening all isolates was employed (total reaction volume 1 mL), 50 of the isolates were found to be capable of SNPs generation, with two species showing particularly high activity, one of the division Ascomycota (Aspergillus oryzae (Ahlb.) Cohn) and the other one of the division Deuteromycota (Fusarium solani (Mart.) Sacc.). Since little work has been previously published on extracellular SNPs production by F. solani, the factors impacting SNPs production by this isolate were further studied. The ITS sequence confirmed the identity of the isolate as F. solani (GenBank accession number: MH005062). Fungal filtrate of four-day cultures was treated with 1 mM AgNO3 which resulted in SNPs production as detected using UV-Vis spectroscopy, and characterized by transmission electron microscope (TEM), particles were spherical in shape with an average size of 22.6 ±1.2 nm. Selected area electron diffraction (SAED) spectrum confirmed presence of metallic silver. The antibacterial effect of synthesized SNPs against Bacillus cereus SBTBC, Enterococcus faecalis 8J, Listeria monocytogenes 10403S, Staphylococcus aureus 7A, Escherichia coli and Salmonilla was studied using microplate dilution method. The half maximal inhibitory concentration (EC50) against B. cereus SBTBC, E. faecalis 8J, E. coli and Salmonilla were 9.2, 6.2, 2.6 and 0.9 mg/mL, respectively. Whereas, monocytogenes 10403S and S. aureus 7A were found to be resistant to the SNPs inhibitory effect of SNPs at all tested concentrations. Silver nanoparticles generation was optimized using different parameters, including reaction pH (pH 8), longer reaction time (up to 100 h), absence of oxygen, mycelium levels at 0.1 g/mL, use of YMPG medium, high temperature, undiluted fungal filtrate and AgNO3 concentration at 1.5 mM, all resulted in better SNPs generation. Our findings demonstrate that filtrates of a novel F. solani marine isolate are effective in SNPs generation and presented a further understanding of processes impacting SNPs production that resulted in increased yields. On the other hand, another fungus with an economic importance was selected to test the efficiency of studied optimized conditions from another fungal group (Ascomycota). Also, we demonstrated the synthesis of SNPs using fungal filtrate of A. oryzae, with an average particles size of 12.31 ±0.62 nm under standard conditions. The antibacterial effect was studied with the same method as previously mentioned, using the same bacterial strains. The EC50 of B. cereus SBTBC, E. faecalis 8J, E. coli and Salmonilla were 1.9, 1.6, 0.3 and 0.2 mg/mL, respectively. Also, L. monocytogenes 10403S and S. aureus 7A were resistant to the inhibitory effect of generated SNPs at all tested concentrations. The One-way ANOVA statistical analysis showed that there is a significant difference comparing the antibacterial effect of different size of SNPs generated by the two fungal isolates, against B. cereus SBTBC, E. faecalis 8J, E. coli and Salmonilla, but non-significant difference was found in case of the two resistant strains. Nearly, all optimized conditions for SNPs generation using filtrates of F. solani worked successfully with generating SNPs using filtrates of A. oryzae, except for the pH (pH 9). Extracellular protein profile of A. oryzae was identified using MALDI/TOF Mass spectrometry to determine reductase protein(s) in the fungal filtrate that are responsible for SNPs formation. FAD dependent oxidoreductase was one of the eight most prominent protein bands. We suggest that this protein plays an essential role in converting silver ions (Ag+) into SNPs. as it was the only identified protein with a reductase activity in the fungal filtrate. Our findings suggest that this species would be useful for large scale and stabilized SNPs production.