Antimicrobial resistance is a global challenge exacerbated by antibiotic misuse and inadequate infection control. This study explores the effects of antibiotics in semen extenders on the vaginal bacteria of the mare and investigates the separation of spermatozoa from bacteria in seminal plasma as an alternative to adding antibiotics to semen extenders. In addition, it examines the diversity of the seminal microbiota in healthy stallions across different countries. Antimicrobial resistance was influenced by breeding status and age of the mare, as well as environmental factors such as housing and location, and exposure to antibiotics in semen extenders. There were geographic differences in the diversity of the seminal microbiota, with implications for tailored antibiotic use. While phylum level composition of seminal microbiota remains consistent, genus level variations may stem from husbandry or individual differences. Bacterial load could be reduced by Single Layer Centrifugation with a low density colloid, without compromising sperm quality. This method has potential for circumventing the need for antibiotics in semen extenders, although further research is needed to confirm sperm fertilizing capacity