Trichoderma hamatum (Bonord.) Bainier was evaluated for its antagonistic potential against Fusarium oxysporum Schlecht. emend. Snyder and Hansen sp. lentis, the causal agent of vascular wilt disease of lentil (Lens culinaris Medikus). Hyphal interactions on Petri plates resulted in an increase in the number of conidial spores and an increase in the vegetative growth of T. hamatum, and a decrease in the hyphal formation and sporulation of F. oxysporum f. sp. lentis. Electron and light microscopical observations suggested that hyphae of T. hamatum established aggressive contact and attachment with the hyphae of the pathogen. Growing in parallel, coiled densely and tightly, T. hamatum may penetrate those of the pathogen hyphae causing collapse due to the loss of turgor pressure. The cellulolytic enzymes produced by T. hamatum presented sufficient characteristics for its antifungal activity in the hyphae hydrolysis and competition process. In growth room and glasshouse experiments, the addition of the conidial suspension of T. hamatum or its culture filtrate to soil, significantly (p ≤ 0.05) reduced development and spore germination of F. oxysporum. In the rhizosphere, T. hamatum occupied the same ecological niches (rhizosphere, roots, and stems) parasitizing F. oxysporum f. sp. lentis. Treatments using T. hamatum delayed the time of infection by F. oxysporum, promoted the growth, and increased the dry weight of a susceptible variety of lentil (cv. Precoz). The percent of mortality was reduced to 33 and 40% when using T. hamatum and its filtrate, respectively, compared to 93% in the control treatment during the 65 days of growing in loam/sand (2:1 vol/vol) under glasshouse conditions. Plant colonization results indicate that T. hamatum and its filtrate significantly (p ≤ 0.05) reduced development of the pathogen in the vascular tissue of lentil to < 30 and < 40% stem colonization, respectively, compared to 100% in the plant pathogen control. Our results suggest that potential biocontrol mechanisms of T. hamatum towards F. oxysporum f. sp. lentis were antibiosis by production of antifungal enzymes, complex mechanisms of mycoparasitism, competition for key nutrients and/or ecological niches, growth promotion, and a combination of these effects. This study results hold important suggestions for further development of effective strategies of the biological control of Fusarium vascular wilt of lentil.