Increased emergence in microbial resistance to antibiotics is a growing problem globally. A retrospective investigation was made of antimicrobial resistance in a total of 2,345 Escherichia coli isolates from clinical cases submitted to Regional Veterinary Laboratory of Bukit Tengah, Penang, Malaysia between January 2000 and December 2015. Analysis using WHONET 5.6 revealed that resistance to ampicillin, norfloxacin and aminoglycosides was detected in isolates from poultry and pigs more often than ruminants. Resistant to colistin was higher in ruminants compared to other animal groups. Nor floxacin susceptibility was very high in isolates from ruminants, pet birds (82.2%) and zoo animals (88.6%). Isolates from pet birds had higher resistant rate to all of the antimicrobials tested compared to zoo and companion animals. Multidrug-resistant (MDR) E. coli isolates (29.25%, 684/2345) were detected in this study. Further efforts, for instance, national monitoring and surveillance, are required to quantify the exposure of animals to antimicrobial agents and resistant pathogenic or commensal bacteria in the animals and also its environment. Such useful information indeed will assist the prudent use of antimicrobial agents in veterinary practice; therefore, suppress the emergence of antimicrobial resistance in animals.