The metabolites of the fungal strain Rhizopus oryaze were used as a biocatalyst for the green-synthesis of magnesium oxide nanoparticles (MgO-NPs). The production methodology was optimized to attain the maximum productivity as follows: 4 mM of precursor, at pH 8, incubation temperature of 35 °C, and reaction time of 36 h between metabolites and precursor. The as-formed MgO-NPs were characterized by UV-Vis spectroscopy, TEM, SEM-EDX, XRD, DLS, FT-IR, and XPS analyses. These analytical techniques proved to gain crystalline, homogenous, and well-dispersed spherical MgO-NPs with an average size of 20.38 ± 9.9 nm. The potentiality of MgO-NPs was dose- and time-dependent. The biogenic MgO-NPs was found to be a promising antimicrobial agent against the pathogens including Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans with inhibition zones of 10.6 ± 0.4, 11.5 ± 0.5, 13.7 ± 0.5, 14.3 ± 0.7, and 14.7 ± 0.6 mm, respectively, at 200 μg mL–1. Moreover, MgO-NPs manifested larvicidal and adult repellence activity against Culex pipiens at very low concentrations. The highest decolorization percentages of tanning effluents were 95.6 ± 1.6% at 100 µg/ 100 mL after 180 min. At this condition, the physicochemical parameters of tannery effluents, including TSS, TDS, BOD, COD, and conductivity were reduced with percentages of 97.9%, 98.2%, 87.8%, 95.9%, and 97.3%, respectively. Moreover, the chromium ion was adsorbed with percentages of 98.2% at optimum experimental conditions.