NANOMATERIALS have received much attention in the last two decades and have been introduced into most life fields, especially the medical field. Therefore, the present research aims to biosynthesize titanium dioxide nanoparticles (TiO2NPs) by Aspergillus niger DS22(ON076463.1) and evaluate its antibacterial, anti-inflammatory, and wound-healing activities. Forty-five fungal isolates were isolated from nine different Egyptian soil samples and tested for producing TiO2 nanoparticles (NPs). The most potent fungal isolate that produced the promising potential TiO2NPs was identified by 18S ribosomal RNA as Aspergillus niger DS22. The factors affecting the secretion of bioactive compounds assisted in TiO2 nanoparticle biosynthesis were optimized. Gas chromatography-mass spectrometry (GC–MS) and High-performance liquid chromatography (HPLC) analysis of Aspergillus niger DS22(ON076463.1) filtrate showed the existence of various active metabolites, which exhibited antibacterial and anti-inflammatory impacts. Results showed that TiO2NPs were elliptical to spherical, ranging in size from 10.4 to 45.8 nm with a size mean of 26.619±7.577nm. TiO2NPs had antibacterial action versus five pathogenic bacterial strains. The maximum activity was against Methicillin-Resistant Staphylococcus aureus MRSA(ATCC 25923) (34mm inhibition zone) and the minimum activity was against Klebsiella pneumonia(ATCC 700603) (19.66mm inhibition zone). Also, TiO2NPs exhibited inhibition percentages of human erythrocyte hemolysis (97.7%) at 1000μg/mL concentration compared with standard Indomethacin (98.6%). Besides, TiO2NPs were effective in human dermal cells wound recovery after 48h by (65.96%) linked with control (58.49%). These findings evoked that TiO2NPs have promising antibacterial, wound-healing, and anti-inflammatory actions, which may be an auspicious approach for pharmaceutical applications.