In the current study, zinc oxide nanoparticles (ZnONPs) were successfully mycosynthesized using Rhizopus arrhizus NWMA1 and formulated into carboxymethyl cellulose (CMC) and starch (ST) as nanocomposite (CMC/St@ZnONPs). The ZnONPs anf formulated CMC/ST@ZnONPs were characterized physiochemically and topographically as well.  ZnONPs UV-visible spectrum presented a peak at 270 nm. Additionally, the Fourier transform infrared (FTIR) of CMC/ST@ZnONPs emphasized the formulation. Moreover, X- ray diffraction (XRD) diffraction of ZnONPs and CMC/ST@ZnONPs were presented in the crystal plane of ZnONPs that decreased after formulation according to the effect of biopolymers. The topographical analysis including transmission electron microscopy (TEM), selected area electronic diffraction (SAED), scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) affirmed the formulation of ZnONPs and incorporation of ZnONPs into CMC/ST@ZnONPs.  The prepared compounds were assessed for antimicrobial activity toward multi-drug resistant bacterial and fungal isolates as well as for anticancer activity toward MCF7 cancerous cell line. Antimicrobial results revealed that CMC/St@ZnONPs nanocomposite showed activity higher than ZnONPs where inhibition zones were 40, 25, 43, 17, 25, 25, 25 and 45 mm against K. pneumonia 124, K. pneumonia 117, B. subtilis, K. pneumonia 115, Acenetobacter bumanii 110, Pseudomonas aerogenosa, E. coli 127, C. albicans. Furthermore, ZnONPs exhibited anticancer activity higher than CMC/St@ZnONPs nanocomposite toward MCF7 cells where IC50 was 61.22 ± 1.09, and 84.47 ± 2.48µg mL^–1 respectively. In conclusion, this study succeeded in the mycosynthesis of ZnONPs and CMC/St@ZnONPs nanocomposite which had promising antimicrobial and anticancer activity.