The main objective of this study was to determine the Burkholderia community structure associated with areas under different agricultural management and to evaluate to which extent this community structure is affected by changes in agricultural management. Two fields with distinct soil history (arable land and permanent grassland) were exposed to three agricultural management regimes (crop rotation, maize monoculture, and grassland). By using a culture-independent approach, based on a Burkholderia-specific polymerase chain reaction–denaturing gradient gel electrophoresis system, it was possible to observe the conversion of Burkholderia communities typical for permanent grassland to those of arable land after four consecutive years. However, the time needed to achieve the reverse transition, i.e., converting the Burkholderia community associated with arable land to that of grassland, was beyond the duration of the field experiment. In addition, by applying principal response curves, the direction and extent of the conversion from grassland to arable land (maize monoculture and to crop rotation) were determined. Hence, the results suggested that agricultural practices, such as fertilization and tillage, were more effective in changing the Burkholderia community structure than agricultural management regime. To determine the effect of agricultural management on the Burkholderia population with biocontrol abilities, the culturable fraction of the Burkholderia community was assessed. The areas under permanent grassland and grassland converted to maize monoculture had the highest percentages of Burkholderia strains with antagonistic activity against Rhizoctonia solani AG-3, mainly Burkholderia pyrrocinia and Burkholderia sp. LMG 22929. The isolation frequency of antagonistic isolates from arable land was extremely low. Our results indicate that (changes in) agricultural management, mainly crop rotation, affect the frequency of isolation of antagonistic Burkholderia strains and that grassland represents a reservoir of Burkholderia species with great potential for agricultural applications.