The biological manufacturing of zinc oxide nanoparticles (ZnO-NPs) using renewable sources is safe, harmless, and compatible with the environment. The capacity of Hypnea pannosa to synthesize ZnO-NPs was investigated in this work. Ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), and zeta potential analysis were employed to characterize the ZnO-NPs. The created ZnO-NPs showed antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Acinetobacter baumannii, Candida albicans, and Candida auris. ZnO-NPs showed an MIC of 12.5 µg/mL against S. aureus, E. faecalis, K. pneumoniae, C. albicans, and C. auris, but it had a 25 µg/mL against Acinetobacter baumannii. ZnO-NPs’ ability to scavenge free radicals was assessed using the 1,1-diphenyl-2-picryl hydrazyl (DPPH) technique with IC50 of 36.2 µg/mL. Anti-inflammatory activity of ZnO-NPs compared to indomethacin at 1000 µg/mL was investigated, where the membrane’s maximum stabilizer was 93.3%. ZnO-NPs demonstrated anticancer activity against PC3 and Caco2 cell lines with IC50 of 174.3 μg/mL and 83.3 μg/mL, respectively. Furthermore, ZnO-NPs demonstrated a range of anti-biofilm activities against Pseudomonas aeruginosa and Staphylococcus aureus. Furthermore, ZnO-NPs showed encouraging antiviral effect versus COX B4 as well as HSV1 with antiviral activities of 54.8% and 61.1%, respectively.