We applied biosynthesized titanium and selenium nanoparticles, prepared using a fungal water extract of Trichoderma harzianum (T. harzianum), to eradicate tomota wilt infection. Transmission electron microscopy (TEM), Scanning electron microscopy (SEM), Energy Dispersive X-Ray analysis, and Transmission electron microscopy/X-ray diffraction (TEM/XRD) techniques were used to characterize the spherical metal nanoparticles, whose diameters were 16.0 nm for selenium nanoparticles (SeNPs) and 50.0 nm for titanium nanoparticles (TiNPs). This confirmed the efficient biosynthesis of the nanoparticles. Under greenhouse conditions, the effectiveness of TiNPs and SeNPs produced by nonpathogenic fungi (T. harzianum) against the pathogen responsible for the tomato wilt disease, Fusarium oxysporum (F. oxysporum), was studied. Based on the results, the most efficient method for combating the pathogen that causes tomato wilt was used in open fields, whereas pot studies were conducted in greenhouse conditions. All tested treatments considerably lowered tomato plant wilt disease in both the greenhouse and the open field. The disease severity was reduced by 20.4% using TiNPs at high concentrations of 150 ppm and by 41.5% using SeNPs. Compared to conventional antibiotics, the antibacterial activity assessment of the biosynthesized TiNPs and SeNPs revealed a significant effect versus pathogenic bacteria and fungi, with a negligible influence on the examined human and animal microflora. The findings showed that biosynthesized TiNPs and SeNPs can be applied to suppress the plant pathogen F. oxysporum in a way that is safe for the microflora of humans and animals. This is the first instance where the nanocidal activity of biological TiNPs and SeNPs has been used against the pathogen that causes tomato wilt.