Salmonella enterica serotype Cerro (S. Cerro) is an emerging Salmonella serotype isolated from cattle, but the association of S. Cerro with disease is not well understood. While comparative genomic analyses of bovine S. Cerro isolates have indicated mutations in elements associated with virulence, the correlation of S. Cerro fecal shedding with clinical disease in cattle varies between epidemiologic studies. The primary objective of this study was to characterize the infection-relevant phenotypes of S. Cerro fecal isolates obtained from neonatal calves born on a dairy farm in Wisconsin, USA. The S. Cerro isolates varied in biofilm production and sensitivity to the bile salt deoxycholate. All S. Cerro isolates were sensitive to sodium hypochlorite, hydrogen peroxide, and acidic shock. However, S. Cerro isolates were resistant to nitric oxide stress. Two S. Cerro isolates were unable to compete with S. Typhimurium during infection of calf ligated intestinal loops, indicating decreased fitness in vivo. Together, our data suggest that S. Cerro is sensitive to some innate antimicrobial defenses present in the gut, many of which are also used to control Salmonella in the environment. The observed phenotypic variation in S. Cerro isolates from a single farm suggest phenotypic plasticity that could impact infectious potential, transmission, and persistence on a farm.IMPORTANCESalmonella enterica is a zoonotic pathogen that threatens both human and animal health. Salmonella enterica serotype Cerro is being isolated from cattle at increasing frequency over the past two decades; however, its association with clinical disease is unclear. The goal of this study was to characterize infection-relevant phenotypes of S. Cerro isolates obtained from dairy calves from a single farm. Our work shows that there can be variation among temporally related S. Cerro isolates and that these isolates are sensitive to killing by toxic compounds of the innate immune system and those used for environmental control of Salmonella. This work contributes to our understanding of the pathogenic potential of the emerging pathogen S. Cerro.