Background: Pyometra is a diestrual chronic disease frequently associated with Escherichia coli. Initial pyometra treatment involves empiric antimicrobial therapy whose suitability should be confirmed by antimicrobial susceptibility testing. Antimicrobial resistance is a major health issue for veterinary medicine, rendering surveillance studies essential. Our goal was to determine the susceptibility profile of E. coli isolates obtained from healthy and pyometra-presenting dogs and to compare the application of different antimicrobial susceptibility guidelines. Methods: The antimicrobial susceptibility profile (ASP) of 74 E. coli isolates was determined by disk diffusion, using six antimicrobials commonly used in veterinary medicine. Profiles were assessed by CLSI VET01S, CLSI M100 and EUCAST guidelines. β-lactamases-encoding genes blaTEM, blaSHV and blaOXA were detected by multiplex PCR. Biofilm production ability was evaluated by pellicle formation assays in Luria–Bertani medium. Results: Variations in the resistance frequency were observed for amoxicillin/clavulanic acid, cephalexin and cefotaxime (29.7–54.1%, 10.8–16.2% and 1.4–4.1%, respectively). Results varied slightly between clinical and commensal isolates, as well as their biofilm-forming ability. Genes blaTEM, blaSHV and blaOXA were detected in 25.5%, 11.8% and 9.8% of isolates, respectively. Conclusions: Results show the importance of ASP determination in veterinary isolates and the need for using standardized and validated testing methods and harmonized interpretive criteria.