Surgical preparation reduces commensal bacterial load. Currently, no standardized preoperative ocular preparation method in the dog has been reported. Previous studies use culture-based methods to determine commensal bacterial populations. Recent reports suggest that high-throughput sequencing may be superior to culture techniques to determine bacterial communities in the eye and other tissues. The goal of this study was to describe the conjunctival commensal ocular microbiome and bacterial community using DNA sequencing and aerobic cultures of six normal, healthy dogs and investigate the short and long-term effects of an antiseptic protocol on the ocular microbiome. Samples were obtained prior to, immediately following, 24 hours following, and 4 weeks following ocular preparation. The Mississippi State University microbiology laboratory evaluated aerobic cultures, and the Gastrointestinal Laboratory of Texas A&M University performed DNA sequencing. This is the first study to show short and long-term effects of standard ocular surgical preparation on the ocular surface microbiome.