Nowadays, membranes and filters made of nanofibers are extensively considered in various fields, such as medicine, water purification, etc. Using an environmentally-friendly method, this study fabricated a nanofiber-based on PVA, bentonite, and silver nanoparticles (AgNPs) with good mechanical strength. Due to conventional AgNPs synthesis methods' toxicity, AgNPs were synthesized by Fusarium sporotrichioides. The MGYP culture medium and the AgNO3 12 M were optimal for Bio-AgNPs synthesis. Bio-AgNPs particle size and zeta potentials were 58 nm and -16.8 mV, respectively. The growth inhibition zone of Bio-AgNPs in E. coli and S. aureus was 0.53 and 0.80 mm, respectively and comparable with chloramphenicol. Then, the optimized Bio-AgNPs and a sample with chemical synthesis AgNPs were used to prepare the PVA/nano-Bentonite/nano-Ag (PVA/NB/AgNPs) nanofiber. The presence of silver in the nanofibers was confirmed through EDX and XRD methods. The diameter of nanofibers was 230 nm. The antibacterial activity of PVA/NB/Bio-AgNPs nanofibers was significantly better against both gram-positive and gram-negative bacteria compared to PVA/NB/Chem-AgNPs nanofibers. The produced membrane is suitable for water treatment, food packaging, and wound dressing because of its good thermal, mechanical, and antibacterial properties.