Display omitted •Purpureocillium lilacinum is an effective entomopathogen of Acromyrmex lundii ants.•Inoculations with 1×106conidiaml−1 were responsible for the death of 85.6% (80.6–89.7) of the ants.•Inoculations with higher concentrations of P. lilacinum showed faster mortality and greater percentage of infection.•P. lilacinum has great capacity to infect ants despite high natural entomopathogen loads.•P. lilacinum was horizontally transmitted from inoculated to non-inoculated ants. Many leaf-cutter ant species are well known pests in Latin America, including species of the genera Acromyrmex and Atta. An environmentally friendly strategy to reduce the number of leafcutter ants and avoid indiscriminate use of chemical pesticides is biological control. In this work we evaluated the effectiveness of a strain of the entomopathogen Purpureocillium lilacinum, against worker ants from six Acromyrmex lundii field colonies, after immersions in pure suspensions at a concentration of 1×106conidiaml−1. Survival of ants treated with P. lilacinum was significantly lower than that recorded in controls, and median lethal time (LT50) was 6–7days. P. lilacinum was responsible for 85.6% (80.6–89.7) of the mortality in inoculated ants, in which we found that the percentage of other entomopathogens that naturally infected ants decreased also, suggesting a good competitive capability of the fungus. Horizontal transmission to non-inoculated ants was also evidenced, given that 58.5% (41.9–64.2) of them died because of P. lilacinum. Moreover, we tested pathogenicity for three concentrations of this strain (1.0×104, 106 and 108conidiaml−1) and found a significantly faster mortality of ants and greater median percentage of infection at 108conidiaml−1 of P. lilacinum. CL50 value was 2.8×105conidiaml−1. We thus propose the use of P. lilacinum as a biological control agent of leafcutter ants in crops and plantations.