Background/objectives: Infections caused by multidrug-resistant (MDR) bacteria are becoming increasingly difficult to treat with recommended antimicrobials. Considering the critical and growing challenge of antimicrobial resistance (AMR), this study aims to evaluate the antimicrobial susceptibility patterns of Escherichia coli clinical isolates from dogs in Grenada. This research project consists of two distinct studies: a retrospective analysis of AMR in canine E. coli isolates collected between 2010 and 2020, and a cross-sectional study characterizing the genotypic AMR profiles of E. coli isolates obtained between April and June 2023. Methods: A retrospective analysis of antibacterial sensitivity test (ABST) reports from canine clinical samples submitted to the Small Animal Clinic at St. George’s University (SGU), St. George’s, Grenada, between 2010 and 2020 revealed a notable prevalence of AMR among canine E. coli isolates. To further investigate the underlying mechanisms of this resistance, the study analyzed canine E. coli isolates that exhibited phenotypic resistance in ABST assays. These isolates were subsequently screened for AMR-associated genes using polymerase chain reaction (PCR) and next-generation sequencing (NGS). Results: The retrospective study identified 153 canine clinical isolates positive for E. coli. The antimicrobial drugs, imipenem, cefotaxime and ciprofloxacin were found to be highly effective against these isolates. However, a gradual increase in AMR was observed for amoxicillin–clavulanic acid (34.88%), ampicillin–sulbactam (17.31%), cephalexin (43.08%), cefpodoxime (22.31%), cephalothin (68.42%), and doxycycline (37.04%). In the prospective study, PCR analysis of resistant canine E. coli isolates detected the tetA (577 bp) and blaTEM (686 bp) genes. These AMR determinants were further confirmed through analysis of NGS reads and assembled contigs. Additionally, NGS-based predictions identified genes associated with resistance to aminoglycosides and potentiated sulfonamides. Conclusions: This study demonstrates that E. coli from dogs in Grenada exhibits resistance to tetracycline and several β-lactam antimicrobials. These findings underscore the need for rational antimicrobial stewardship and continuous AMR surveillance in small animal practice within the region.