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Soilborne pathogens reduce 60% of the yield of onion crops. A common fungal pathogen causing wilt disease and severe losses is Fusarium basal rot (FBR). In this study, the combination of Arbuscular Mycorrhizal Fungi (AMF) with Trichoderma harzianum was investigated against FBR. Onion samples were collected from the Ankara–Polatlı region. Among the isolates, isolate S6 was identified as F. oxysporum f. sp. cepae (FOC) using morphological and molecular methods and pathogenicity tests. Different combinations of AMF (Funneliformis mosseae pure strain and the commercial AMF) and T. harzianum were inoculated on susceptible onion cultivars (Seç, Gence, and Şampiyon). The effects of the treatments on FOC biocontrol were studied under growth chamber conditions. The results showed that Şampiyon was the most resistant, while Gence was the most susceptible to basal rot disease. Different colonization rates (8.91–24%), spore densities (16.4–50.4 spore/10 g soil), and the extent to which a plant needs mycorrhizal conditions to grow to its maximum potential (i.e., mycorrhizal dependencies—18.3–51.9%) were recorded by treatment. Both single and combined applications of AMF and Trichoderma applications suppressed FOC. Suppressive effects were more pronounced when the F. mosseae pure strain was used alone (when F. mosseae was used, disease severity decreased from 90 to 68%, p < 0.05). The F. mosseae pure strain also showed the best plant growth promotion and phosphorus content release. The results indicate an interesting potential use of F. mosseae and the combination of AMF with T. harzianum in the management of FOC in onions. [ABSTRACT FROM AUTHOR]