The present study demonstrates a positive correlation between zinc metal tolerance ability of an isolated fungi and their potential for the synthesis of zinc oxide nanoparticles (ZnO-NPs). A total of 5 fungal cultures were isolated from the rhizospheric soils of plants naturally growing at Sharkia Governorate in Egypt and identified based on morphological characteristics. These isolates are belongs to Aspergillus niger (An), Aspergillus tubulin (At), Aspergillus fumigatus(Af), Penicillium citrinum (Pc) and Fusarium oxysporum (Fo). These isolates were usedin the synthesis of zinc-oxide nanoparticles (An-ZnO-NPs, At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-NPs and Fo- ZnO-NPs) using Zinc sulfate as the precursor compared to the references strains of A. tubingensis Mosseray AUMC No.6915, A. fumigatus Fresenius AUMC No.48and A. terreus Thom AUMC No.75. Aspergillus and Fusarium isolates have been shown to have a high zinc metal tolerance ability and a potential for extracellular synthesis of ZnO nanoparticles under ambient conditions. The synthesized ZnO-NPs were tested by the detection of a notable absorption peak at 285 to 296 nm, appearing in UV–Vis spectra due to surface-plasmon-resonance. Transmission electron microscope (TEM) results revealed that An-ZnO-NPs, At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-NPs and Fo- ZnO-NPs exhibited a crystalline structure with hexagonal wurtzite shape (30–100 nm size). ZnO nanoparticles exhibited excellent antibacterial activity against tested Gram-positive and Gram-negative bacteria. The ZnO nanoparticles showed better antibacterial activity against Staphylococcus. aureus, Listeria. monocytogenes and Bacillus cereus compared to Salmonella enterica, Escherichia coli and Pseudomonas aerogenosa. The effectiveness of inhibition of the microbial biofilms formation of S.aureus, L. monocytogenes and
B. cereus compared to S. enterica, E. coli and P. aerogenosa was analyzed at a concentration of 100 μg/ ml.