A total of 22 fungal isolates were acquired and identified, with these isolates originating from wheat seeds, Sham-6 variety obtained from grain stores in Nineveh Governorate, 2024-2025 season, the fungus Aspergillus niger was the most frequently in wheat seeds with rate (31.81%) followed by Aspergillus terrus (27.27%) and Rhizoctonia solani (18.18%), while A. flavus and Alternaria sp. reached (13.63 %),(9.09 %) Consecutively. as well as the samples (Abaa-99 and Jihan) obtained from local markets were 100% contaminated with the Rhizopus sp. Bio- control strategies including fungal isolates, is an environment friendly strategy that supports the sustainable development goals of reducing the use of chemical pesticides that have a harmful effect on human health in the long term.The antagonistic activity assessed in vitro using the dual culture technique and Antibiosis activity by Using non-volatile metabolites to detect antifungal activity of Trichoderma harzianum against Rhizoctonia solani that showed statistically significant inhibition which reach scale 1 for dual culture technique and The inhibition percentage was reached 55.5% for Antibiosis activity.The morphological identification for the chosen pathogenic fungus R.solani confirmed by using polymerase chain reaction assay of ITS1, 5.8S, and ITS2 are parts of the internal transcribed spacer of the rDNA, The nucleotide sequences deposited in the Gene Bank. Based on the lineup process results, the local fungal isolate that was chosen had a nucleotide sequence that was of the type R. solani with the PV138271 registration number.