Sampling was conducted at The Ohio State University's Veterinary Teaching Hospital (OSU-VTH) to evaluate the extent of environmental contamination with Salmonella enterica, at 1-week intervals beginning March 19, 2007, through May 21, 2007. Environmental samples were collected from various surface and floor locations in the equine and food animal areas using sterile moistened gauze sponges. All samples were processed using standard bacteriologic culture to identify the presence of Salmonella spp. Genetic relatedness of isolates was assessed using amplified fragment length polymorphism (AFLP) procedures, and minimum inhibitory concentrations to a panel of antimicrobial drugs were determined using microbroth dilution. A total of 16 Salmonella isolates were recovered from 270 (5.9%) cultured environmental and animal samples, with prevalence ranging from 0% to 32% on individual sampling dates. A total of 9% of the samples from the food animal section and 2.5% of the samples from the equine section tested positive for Salmonella (p = 0.03). The 16 isolates represented seven different clonal strains and four different serotypes (Paratyphi B var. L-tartate n = 3, Kentucky n = 5, Cerro n = 7, Montevideo n = 1), most of which were pansusceptible to a panel of antimicrobial drugs. Our results indicate that animal treatment areas with a high population of animals or cases within the veterinary teaching hospital can become contaminated with Salmonella, especially in high traffic areas that may facilitate horizontal dissemination. The most common occurrence appears to be infected agricultural animals that contaminate the hospital environment, but normal cleaning and disinfection appears to effectively prevent long-term contamination.