Neuro- and nephrotoxicity of polymyxins are known but clinical studies in horses are lacking. The aim of this study was to describe neurogenic and nephrogenic side effects of hospitalized horses receiving Polymyxin B (PolyB) as part of their treatment plan. Twenty horses diagnosed with surgical colic (n= 11), peritonitis (n= 5), typhlocolitis (n= 2), pneumonia, and pyometra (eachn= 1) were included. Antimicrobial treatment was randomized to GENTA (gentamicin 10 mg/kg bwt q24 h IV, penicillin 30.000 IU/kg q6 h IV) or NO GENTA (marbofloxacin 2 mg/kg bwt q24 h IV, penicillin 30.000 IU/kg q6 h IV). The duration of PolyB treatment ranged from 1 to 4 days. Clinical and neurological examinations were performed, and serum PolyB concentrations were measured daily during and three days following PolyB treatment. Urinary analysis, plasma creatinine, urea and SDMA were assessed every other day. Video recordings of neurological examinations were graded by three blinded observers. All horses showed ataxia during PolyB treatment in both groups (median maximum ataxia score of 3/5, range 1–3/5). Weakness was detected in 15/20 (75%) horses. In 8/14 horses, the urinary γ-glutamyltransferase (GGT)/creatinine ratio was elevated. Plasma creatinine was mildly elevated in 1/16 horses, and SDMA in 2/10 horses. Mixed-model analysis showed a significant effect of time since last PolyB dose (p= 0.0001, proportional odds: 0.94) on the ataxia score. Ataxia and weakness should be considered as reversible adverse effects in hospitalized horses receiving PolyB. Signs of tubular damage occurred in a considerable number of horses; therefore, the nephrotoxic effect of polymyxins should be considered and urinary function monitored.