Cyanobacteria, being a prominent category of phototrophic organism, exhibit substantial potential as a valuable source of bioactive compounds and phytonutrients, including liposomes, amino derivatives, proteins, and carotenoids. In this investigation, a polyphasic approach was employed to isolate and characterize a newly discovered cyanobacterial strain from a rice field in the Garh Moor district of Jhang. Desertifilum sp. TN-15, a unique and less explored cyanobacterial strain, holds significant promise as a novel candidate for the synthesis of nanoparticles. This noticeable research gap underscores the novelty and untapped potential of Desertifilum sp. TN-15 in the field of nanomedicine. The characterization of the biogenically synthesized ZnO-NPs involved the application of diverse analytical techniques. Ultraviolet-visible spectroscopy revealed a surface plasmon resonance peak at 298 nm. Fourier transform infrared spectral analysis was utilized to confirm the involvement of biomolecules in the biogenic synthesis and stability. Scanning electron microscopy was employed to probe the surface morphology of the biogenic ZnO-NPs unveiling their size of 94.80 nm and star-shaped. Furthermore, X-ray diffraction analysis substantiated the crystalline nature of ZnO-NPs, with a crystalline size measuring 46 nm. To assess the physical stability of ZnO-NPs, zeta potential and dynamic light scattering measurements were conducted, yielding values of + 31.6 mV, and 94.80 nm, respectively, indicative of favorable stability. The antibacterial capabilities of Desertifilum sp. TN-15 are attributed to its abundance of bioactive components, including proteins, liposomes, amino derivatives, and carotenoids. Through the synthesis of zinc oxide nanoparticles (ZnO-NPs) with this strain, we have effectively used these chemicals to generate nanoparticles that exhibit noteworthy antibacterial activity against Staphylococcus aureus (MIC: 30.05 ± 0.003 µg/ml). Additionally, the ZnO-NPs displayed potent antifungal activity and antioxidant properties, as well as significant antihemolytic effects on red blood cells (IC50: 4.8 µg/ml). Cytotoxicity assessment using brine shrimps revealed an IC50 value of 3.1 µg/ml. The multifaceted actions of the biogenically synthesized ZnO-NPs underscore their potential applications in pharmacological and therapeutic fields. This study proposes a novel method for ZnO-NPs production utilizing the recently identified cyanobacterial strain Desertifilum sp. TN-15, highlighting the growing significance of biological systems in the environmentally friendly fabrication of metallic oxide nanomaterials.