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In this study, a ZnO/MnO nanocomposite was myco-fabricated using the isolated endophytic Clonostachys rosea strain EG99 as the nano-factory. The extract of strain EG99, a reducing/capping agent, was successfully titrated with equal quantities of Zn(NO3)2·6H2O and Mn(NO3)2·6H2O (precursors) in a single step to fabricate the rod-shaped ZnO/MnO nanocomposite of size 6.22 nm. The ZnO/MnO nanocomposite was myco-fabricated in 20 min, and the results were validated at 350 and 400 nm using UV–Vis spectroscopy. In a 7-L bioreactor, an industrial biotechnological approach was used to scale up the biomass of this strain, EG99, and the yield of the myco-fabricated ZnO/MnO nanocomposite. A controlled fed-batch fermentation system with a specific nitrogen/carbon ratio and an identical feeding schedule was used in this production process. Higher yields were obtained by adopting a controlled fed-batch fermentation approach in a 7-L bioreactor with a regular feeding schedule using a nitrogen/carbon ratio of 1:200. Overall, the fed-batch produced 89.2 g/l of biomass at its maximum, 2.44 times more than the batch's 36.51 g/l output. Furthermore, the fed-batch's maximum ZnO/MnO nanocomposite yield was 79.81 g/l, a noteworthy 14.5-fold increase over the batch's yield of 5.52 g/l. Finally, we designed an innovative approach to manage the growth of the endophytic strain EG99 using a controlled fed-batch fermentation mode, supporting the rapid, cheap and eco-friendly myco-fabrication of ZnO/MnO nanocomposite. At a dose of 210 µg/ml, the tested myco-fabricated ZnO/MnO nanocomposite exhibited the maximum antibacterial activity against Staphylococcus aureus (98.31 ± 0.8%), Escherichia coli (96.70 ± 3.29%), and Candida albicans (95.72 ± 0.95%). At the same dose, Staphylococcus aureus biofilm was eradicated in 48 h; however, Escherichia coli and Candida albicans biofilms needed 72 and 96 h, respectively. Our myco-fabricated ZnO/MnO nanocomposite showed strong and highly selective antagonistic effects against a variety of multidrug-resistant human pathogens. Therefore, in upcoming generations of antibiotics, it might be employed as a nano-antibiotic. [ABSTRACT FROM AUTHOR]