Establishing their initial colony is probably the most critical moment in the life of leaf-cutting ants. The non-establishment is connected to abiotic and biotic factors, and the high mortality rates of initial colonies are possibly associated with entomopathogenic or antagonistic microorganisms to the symbiotic fungus present in the soil, hosted by these ants. Fire in the vegetation, depending on the intensity, is known to cause significant changes to the soil physical, chemical, and microbiological properties. The impact of a fire in savannah vegetation (Cerrado) on the establishment of early colonies of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) was evaluated. For this end, two areas were selected, one where there had been an accidental fire, and a contiguous one with the same size and vegetation characteristics without burning. In these areas and in soil collected in the same areas and stored in the laboratory, females recently fertilized in the nuptial flight were placed to excavate the soil and establish their colonies. Post-fire changes in the soil chemical and microbiological properties were quantified and correlated successfully in the establishment of new colonies of this leaf-cutting ant. Under field conditions, the females of A. sexdens rubropilosa did not show preference for which areas to excavate: the ones that had been burned or the ones that were unburned; under this condition, no colony survived according to the evaluation performed 120 d after the nuptial flight. Under laboratory conditions, the majority of the females excavated the soil, whether it had been burned or not. However, the establishment of initial colonies was significantly higher in soils collected far from the surface and in areas that had not directly been affected by the fire, showing a negative effect of fire on colony establishment under laboratory conditions.