Sand bedding material is frequently used in dairy operations to reduce the occurrence of mastitis and enhance cow comfort. One objective of this work was to determine if sand-based bedding also supported the microbiologically based suppression of an introduced bacterial pathogen. Bedding samples were collected in summer, fall, and winter from various locations within a dairy operation and tested for their ability to suppress introduced populations ofEscherichia coliO157:H7. All sources of bedding displayed a heat-sensitive suppressiveness to the pathogen. Differences in suppressiveness were also noted between different samples at room temperature. At just 1 day postinoculation (dpi), the recycled sand bedding catalyzed up to a 1,000-fold reduction inE. colicounts, typically 10-fold greater than the reduction achieved with other substrates, depending on the sampling date. All bedding substrates were able to reduceE. colipopulations by over 10,000-fold within 7 to 15 dpi, regardless of sampling date. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to identify bacterial populations potentially associated with the noted suppression ofE. coliO157:H7 in sand bedding. Eleven terminal restriction fragments (TRFs) were overrepresented in paired comparisons of suppressive and nonsuppressive specimens at multiple sampling points, indicating that they may represent environmentally stable populations of pathogen-suppressing bacteria. Cloning and sequencing of these TRFs indicated that they represent a diverse subset of bacteria, belonging to theCytophaga-Flexibacter-Bacteroidetes,Gammaproteobacteria, andFirmicutes, only a few of which have previously been identified in livestock manure. Such data indicate that microbial suppression may be harnessed to develop new options for mitigating the risk and dispersal of zoonotic bacterial pathogens on dairy farms.