The antibacterial, antifungal, antiviral, antioxidant, and antitumor properties of biosynthesized titanium nanoparticles from isolated and identified Saccharopolyspora spinosa were tested in vitro. Investigation of ultrastructural changes in microbes treated by TiO2 nanoparticles. Elucidation of the role of titanium nanoparticles in enhancing the apoptotic rate of tumour cells and ensuring their minimal cytotoxicity. Actinomycete was isolated from animal compost specimen in starch nitrate agar medium and identified using cultural and morphological features. Actinomycete was tested to synthesize TiO2 nanoparticles where S. spinosa were the most successful isolate to produce nanoparticles and its identification has been done by genetic tools and deposited in genebank. Titanium dioxide nanoparticles were characterized by UV, XRD, EDX, FTIR, TEM, particle size and zeta potential. Antioxidant action of TiO2NPs was evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Antitumor action of TiO2NPs versus A549 cells. Apoptotic rate was evaluated using flow cytometry. Antibacterial, antifungal and antiviral roles of TiO2NPs were determined. The alteration in the most affected microorganisms were examined using transmission electron microscope. S. spinosa was isolated and identified using various morphological and molecular tools and deposited in gene bank with accession number (OP315279.1). White suspension of TiO2 nanoparticles could be seen with spherical shape. TiO2 nanoparticles has promising antioxidant impact with IC50 = 78.35 ± 2.59 µg/ml, as well as antitumor action versus A542 cells with IC50= 96.12± 3.64 upon acceleration of apoptotic rate. TiO2 nanoparticles has minimal toxicity versus Vero cells with CC50 = 402.94± 14.76 µg/ml. TiO2 nanoparticles has promising antimicrobial impact versus Proteus vulgaris, Candida albicans and Heptitis A virus which confirmed by ultrastructure examination. S. spinosa have the capability to produce TiO2 nanoparticles with pleotropic biomedical applications to be verified by in vivo studies.