Background: Carnation, a major cut flower product cultivated economically in Iran, faces economic challenges due to the devastatingFusariumwilt caused byFusarium oxysporumf. sp.dianthi(Fod). To address this issue, twenty-fiveTrichoderma harzianumandT. virideisolates were collected from the rhizosphere soil of three Iranian provinces: Tehran, Markazi, and Fars. RAPD-PCR was applied to analyze the genetic relatedness of the isolates.Results: The RAPD profiles showed genetic diversity among the isolates, with two major clusters. The antagonistic potential of the twenty isolates was evaluated against the carnation wilt caused byFod. The results showed that Th1, Th7, and Th2 isolates ofT. harzianumsignificantly inhibitedFodmycelial growth (58, 56.5, and 48.6%, respectively). AmongT. virideisolates, Tv5, Tv4, and Tv7 exhibited the highest antifungal ability to inhibit mycelial growth. All investigated isolates ofTrichodermasecreted volatile compounds that hinderedFodmycelial growth, with isolates ofT. harzianumranging from 9.3 to 67.5% inhibition and those ofT. viridefrom 25.2 to 50.2%. Additionally, the experiment on competitive saprophytic ability indicated that maximum colonization occurred with Th1, Th7, and Th2 isolates at 78.2, 70.8, and 69.8%, respectively. Lastly, the greenhouse experiment showed a complete pathogen eradication or significant inhibition in the infected carnation afterT. harzianumandT. viride. Conversely, control treatment with theFodpathogen died after 90 days.Conclusions: The investigation suggested thatTrichodermaspp. could be a potential biocontrol agent to mitigateFusariumwilt in carnation and improve production quality, replacing chemical pesticides.