An aqueous extract of Punica granatum peel was used as a biocatalyst for magnesium oxide nanoparticle (MgO-NP) synthesis, which was characterized via UV-Vis spectroscopy, TEM, EDX, FT-IR, XRD, DLS, and zeta potential. Data showed the efficacy of the plant aqueous extract in forming spherical, crystalline-nature, well-arranged MgO-NPs with sizes in the range of 10–45 nm with average sizes of 24.82 ± 8.85 nm. Moreover, EDX analysis revealed that the highest weight and atomic percentages were recorded for Mg and O ions. The green synthesized MgO-NPs showed antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, and Candida albicans in a concentration-dependent manner with clear zones in the range of 8.7 ± 0.6 to 19.7 ± 0.5 mm with various concentrations. Also, the MIC value was varied to be 25 µg mL−1 for Gram-negative bacteria, B. subtilis, and C. albicans and 50 µg mL−1 for S. aureus. Moreover, MgO-NPs showed high activity against the 3rd-instar larvae of Culex quinquefasciatus. The mortality percentages were concentration- and time-dependent. Data analysis showed that the highest mortality was 88.3 ± 3.2%, attained at a concentration of 100 µg mL−1 after 72 h. Also, all originated pupae were malformed and did not hatch to adults, with mortality percentages of 100% at all concentrations. Overall, the P. granatum-mediated MgO-NPs showed promising activity in inhibiting the growth of pathogenic microbes and the hatching of C. quinquefasciatus larvae to adults.