The purposes of this study were to detect non-O157 Shiga toxin-producing Escherichia coli (STEC) in bovine rectums and water in a beef cattle farm in Argentina, and to determine the pathogenic potential of the circulating strains. During the study, 292 rectal swabs from healthy animals and 79 environmental water samples were collected. The rectal swabs and one loop of the Moore swabs, enriched in Escherichia coli broth for 24 h at 37°C, were streaked on MacConkey agar plates and incubated overnight at 37°C. The isolates were characterized by biochemical tests and serotyped. Nonmotile STEC strains were typed for their H-specific (fliC) antigens by polymerase chain reaction (PCR). Isolates were characterized by detection of stx1, stx2, and their variants, eae, ehxA, and saa genes. Macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE) was performed using the PulseNet standardized protocol. From 371 samples analyzed, 36.6% of rectal swabs and 34.2% of water samples were non-O157 STEC-positive by PCR, and 110 strains from rectal swabs, but only three from water, were isolated. The strains were grouped into 24 different serotypes, from which, O103:[H2] (n = 12), O136:H12 (n = 8), O178:H19 (n = 8), and O103:NM (n = 5) were most prevalent, representing 29.2% of the isolates. Predominant genotypes were stx1/eae/ehxA (16.8%) and stx2/saa/ehxA (15.9%). PFGE analysis revealed 56 different patterns, with 65 strains grouped in 19 clusters of 100% similarity. Two STEC O124:H19 strains isolated from rectal swabs and water with a 5-month interval harbored the stx1/stx2/saa/ehxA genotype, and showed an indistinguishable PFGE profile. By comparison, some XbaI-PFGE patterns identified in the present study were identical to the profiles of strains isolated from human, food, and animal sources included in the Argentine PulseNet database. By PCR, similar non-O157 detection rates were found in rectal swabs and water. However, the methodology for water samples needs to be improved, since only three strains from the total number of positive samples were recovered.