Background Anthracnose is a soil-borne tomato (Solanum lycopersicum L.) disease caused by Colletotrichum coccodes and associated with early dying syndrome and postharvest fruits decays leading to significant yield losses. A biocontrol trial was conducted using Penicillium sp. and Gliocladium spp. isolates recovered from soil and compost for the evaluation of their antifungal potential against the target pathogen. Results Cell-free culture filtrates of the microbial agents tested at different concentrations displayed an important antifungal activity based on pathogen mycelial growth inhibition and spore germination suppression. The highest antifungal potential was induced by Penicillium sp. CH6, G. catenulatum Gc1 and G. virens Gv1 cell-free filtrates applied at 3 concentrations (10, 15 and 20% v/v). C. coccodes mycelial growth was significantly reduced by 16 to 84% with chloroform and ethyl acetate extracts of Penicillium sp. CH6 and G. catenulatum Gc1 isolates applied at the concentrations 1, 2.5 and 5% v/v. These treatments had induced an important morphological alteration to pathogen mycelium expressed mainly by a decrease in colony melanization. Testing on pathogen-inoculated fruits, treatment based on cell-free culture filtrates and organic extracts had significantly reduced anthracnose severity as compared to control. Rot lesion diameter was significantly decreased about 22% following treatments with Penicillium sp. CH6 and G. catenulatum Gc1 cell-free filtrates. All chloroform and ethyl acetate extracts of isolates tested had suppressed disease severity by 11 to 59% and 14 to 85%, respectively, as compared to the untreated control. Conclusions The results demonstrated the involvement of bioactive compounds in the cultural filtrates and organic extracts tested. These microbial agents may be explored as an alternative tool for the protection of tomato fruits from fungi-associated decays.