The fungus Fusarium circinatum Nirenberg & O’Donnell causes pine pitch canker, an important disease for conifers worldwide. F. circinatum was first detected in Chile in 2001 and to date is present in nurseries and clonal hedges from Libertador General Bernardo O’Higgins Region to Los Rios Region. The purpose of this study was to evaluate the potential of Trichoderma spp. and Clonostachys spp. strains to control F. circinatum in Pinus radiata D. Don seedlings in the absence of other effective control methods. Eighty-one Trichoderma spp. and Clonostachys spp. strains were evaluated through in vitro assays to determine their ability to act as antagonists of F. circinatum and 21 strains were tested for their ability to reduce post-emergence mortality and increase P. radiata survival under greenhouse conditions. During in vitro experiments, 15 strains of Trichoderma inhibited mycelial growth of the pathogen by more than 60% and one strain of Clonostachys showed parasitism of F. circinatum hyphae. Greenhouse experiments showed no control of the disease when the antagonists were added to substrate after the pathogen. However, when the antagonists were added before the pathogen, four strains (Clonostachys UDC-32 and UDC-222 and Trichoderma UDC-23 and UDC-408) reduced post-emergence mortality between 80 and 100%. Among these strains, only Clonostachys UDC-222 significantly increased the survival of P. radiata seedlings. These results showed that Clonostachys UDC-222 has the potential to be used as a biocontrol agent against F. circinatum in the production of P. radiata plants.