Inundative mass application of Metarhizium brunneum BIPESCO 5 (Hypocreales, Clavicipitaceae) is used for the biological control of Diabrotica v. virgifera (Coleoptera, Chrysomelidae). Long-term field trials were performed in three Austrian maize fields—with different cultivation techniques and infestation rates—in order to evaluate the efficacy of the treatment to control the pest larvae. In addition, the indigenous Metarhizium spp. population structure was assessed to compare the different field sites with BIPESCO 5 mass application. Annual application of the product Granmet-PTM (Metarhizium colonized barley kernels) significantly increased the density of Metarhizium spp. in the treated soil above the upper natural background level of 1000 colony forming units per gram dry weight soil. Although a decrease in the pest population over time was not achieved in heavily infested areas, less damage occurred in treated field sites in comparison to control sites. The Metarhizium population structure was significantly different between the treated field sites. Results showed that inundative mass application should be repeated regularly to achieve good persistence of the biological control agent, and indicated that despite intensive applications, indigenous populations of Metarhizium spp. can coexist in these habitats. To date, crop rotation remains the method of choice for pest reduction in Europe, however continuous and preventive application of M. brunneum may also present an alternative for the successful biological control of Diabrotica.