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Simple Summary: The escalating threat of antimicrobial resistance, identified by the World Health Organization as a top global public health concern, prompts a critical examination of practices contributing to this problem, as in the case of artificial insemination where antimicrobials are commonly included in sperm doses. This study assessed three equine semen processing techniques (Simple Centrifugation, Single-Layer Colloidal Centrifugation, and Filtration) for their impact on quality and microbial load, both immediately after processing and after refrigeration for 48 h. Results showed no significant differences in sperm quality immediately after processing among protocols, except for a higher straightness index in the filtrated and colloidal-centrifuged samples compared to raw semen. In 48 h chilled samples, only the linearity and oscillation index were significantly higher in colloidal-centrifuged samples. Microbial load analysis revealed no significant differences between protocols after refrigeration and minor differences between some protocols and raw sperm values. Thus, irrespective of antibiotic presence, the evaluated methods maintained semen quality and reduced microbial load to the same extent as the traditional antibiotic-containing protocol. These findings suggest the potential of alternative processing protocols, coupled with hygiene practices, to mitigate or eliminate non-therapeutic antibiotic use and contribute to controlling bacterial resistance. Further studies are warranted to validate these results. The study assessed the impact of four equine semen processing techniques on sperm quality and microbial load immediately post-processing and after 48 h of refrigeration. The aim was to explore the potential reduction of prophylactic antibiotic usage in semen extenders. Semen from ten adult stallions was collected and processed under a strict hygiene protocol and divided into four aliquots: Simple Centrifugation with antibiotics (SC+), Simple Centrifugation (SC−), Single-Layer Colloidal Centrifugation (CC−), and Filtration (with SpermFilter®) (F−), all in extenders without antibiotics. Sperm motility, viability, and microbial load on three culture media were assessed. No significant differences were observed in the main in the sperm quality parameters among the four protocols post-processing and at 48 h (p < 0.05 or p < 0.1). Microbial loads in Columbia 5% Sheep Blood Agar and Schaedler vitamin K1 5% Sheep Blood Agar mediums were significantly higher (p < 0.10) for raw semen than for CS+, CC−, and F− post-processing. For Sabouraud Dextrose Agar medium, the microbial load was significantly higher (p < 0.10) in raw semen compared to CS+ and F−. No significant differences (p < 0.10) were found in 48 h chilled samples. Regardless of antibiotic presence, the evaluated processing methods, when combined with rigorous hygiene measures, maintained semen quality and reduced microbial load to the same extent as a traditional protocol using antibiotics. [ABSTRACT FROM AUTHOR]