Objective: Lobesia botrana, the European grapevine moth, affects Vitis vinifera L. and other species of economic importance in a number of countries through damage caused by its larvae in berries and associated secondary diseases such as Botrytis cinerea. Control of the moth in urban areas is difficult due to poor chemical management of infested plants in houses. Additionally, in winter, L. botrana is in its pupal stage covered with a cocoon that prevents the penetration of chemical pesticides. For this reason, the objective of this work was to control the pupal stage with a formulation based on the entomopathogenic fungus Beauveria pseudobassiana in urban areas. Results: The strain RGM 1747 was identified as B. pseudobassiana by multilocus sequence analysis. The biocontrol activity of this formulated fungus against the infestation of vines with breeding pupae without cocoons showed 100% infection 21 days after inoculation under winter conditions. Finally, the biocontrol activity of the formulated fungus against natural infestations of L. botrana in winter in urban areas reached an efficacy of 51%. This result suggests that the B. pseudobassiana formulation is able to penetrate the cocoon and contributes to the integrated pest management of L. botrana.