Horses are hindgut fermenters that harbor a complex intestinal microbiota (IM) which provides key enzymes aiding in the breakdown of complex carbohydrates present in their herbivorous diet. Therefore, these animals are deeply dependent on their IM for digestion and nutrition. Consequently, IM imbalances may result in alteration of fermentation patterns with impact on the animal health and the risk of disease. In this context, strategies for assisting the maintenance of a healthy IM in horses are of interest. However, there is limited research concerning the use of probiotics to improve hindgut fermentation and diet digestibility, with very few studies focusing on the use of lactobacilli strains from equine origin. Herein, we conducted independent fecal batch fermentations, using feces from "Asturcón" horses as inocula, added individually with four different lactobacilli strains (two strains of Lactobacillus acidophilus and two of Ligilactobacillus equi) isolated from this same horse breed. The impact on the gut microbiota composition was assessed by 16S rRNA gene profiling and the metabolic activity (production of short-chain fatty acids) by gas chromatography. The functionality of the lactobacilli strain was determined by monitoring in real-time gas production and determining changes in pH along incubation. L. acidophilus IPLA20127, promoted an increase in IM diversity and in the relative abundance of Lactobacillus genus, as well as higher butyric and valeric acid levels. This strain shows potential as probiotic supplement, without triggering acidification, nor promoting an increase of gas production or abrupt IM changes in our experimental model.