Recently, applying industrial bio-processing strategies are being a promised tool that used to produce large quantities of bioactive metabolites from different microbial cells. So in this study, the statistical experimental designs (Plackett–Burman and Box–Behnken) were used for optimization of local S. violaceoruber SYA3 biomass production (18g/l) and fabrication the highest nano-ZnO mass weight (15g/l). Since the nano-ZnO was fabricated by using the extracted periplasmic metabolites as a reducing agent and Zn(NO3)2.6 H2O as a precursor. The morphological properties and size of the nano-ZnO were evaluated using SEM and TEM analysis. Large-scale production was studied via batch fermentation mode using semi-industrial scale bioreactor (8.9L). The biomass production and the fabricated nano-ZnO was (22.8g/l) and (56.8g/l), respectively. Finally, this work reported developing a new cheap and eco-friendly nano-ZnO fabrication method and applying nano-ZnO as a strong anti-phytopathogens agent.