Abstract Background The leafcutter ant ( Atta cephalotes ) is associated with losses in the agricultural sector, due to its defoliating activity; for its control, biological, mechanical and chemical methods have been developed, the latter associated with adverse effects on human and environmental health. This research validated in the field for the control of the leafcutter ant ( A. cephalotes ) using a mixture of Beauveria bassiana and Trichoderma lignorum spores. Methods The effectiveness from the combination of spores of B. bassiana and T. lignorum with an initial concentration of 2 × 10 9 spores/ml, in the following proportions of B. bassiana and T. lignorum , A (1:1), of each fungus. It was evaluated within the university campus, comparing it with two commercial formulations, Mycotrol ( B. bassiana ) and Mycobac ( T. lignorum ). Additionally, this formulation was evaluated in 49 nests distributed 16 in 14 locations in Colombia. The formulation application was carried out by direct application, using a pump at a speed of 10 ml/m 2 . The effectiveness was estimated from the reduction of the flow of ants, evaluating the statistically significant differences using the ANOVA and Tukey-test. Results Effective control of 90% of the nests was observed in the field phase in 60 days, except in nests with areas > 50 m 2 that were located in regions with high rainfall (annual average precipitation above 7000 mm), such as Buenaventura. Conclusions In this work, it was demonstrated that the combination of B. bassiana and T. lignorum spores represent a viable alternative for the control of the leafcutter ant, in which the effectiveness is related to several factors, including the size of the nest and the rainfall in the area.