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Abstract Free faecal liquid (FFL) is a condition in horses which manifests as differential defecation of solid and liquid phases of faeces. The etiology of FFL is currently unknown, but deviances in the hindgut microbiota has been suggested to be of importance. The present study aimed to compare the faecal bacterial composition of farm-matched horses with (case, n = 50) and without (control, n = 50) FFL. Samples were collected at three different occasions. The V3 and V4 regions of the 16S rRNA gene were amplified and sequenced using Illumina sequencing. Also, samples were cultivated for detection of Clostridioides difficile and Clostridium perfringens. Analysis revealed similar faecal bacterial composition between case and control horses, but an effect of sampling period (p = 0.0001). Within sampling periods, 14 genera were present in higher or lower proportions in case compared to control horses in at least one sampling period. Compared to controls, case horses had higher relative abundance of Alloprevotella (adjusted p