Neutrophil gelatinase-associated lipocalin (NGAL) has been suggested to be a highly sensitive and specific marker of joint infection in humans. The aim of the study was to investigate NGAL concentrations in synovial fluid (SF) from horses with septic synovitis, horses without septic synovitis, and horses with uncertain status. NGAL was measured in 177 admission samples obtained from 152 horses. From a subset of horses (n = 35), additional samples obtained sequentially over the course of treatment were available. Concentrations of NGAL were significantly higher in septic synovitis (n = 47 samples) than in samples classified as non-septic (n = 103) or samples with uncertain status (n = 27), with median NGAL concentrations in the three groups being 1236, 16.8, and 266.4 µg/L, respectively. NGAL discriminated nearly perfectly between septic and non-septic (area under the receiver operating characteristic curve 0.98, 95% confidence interval 0.95–1.00). The optimal cut-off value for maximal sensitivity (87.2%) and specificity (75.0%) to discriminate septic samples from those with uncertain status was 444.6 µg/L, with an area under the receiver operating characteristic curve of 0.85 (95% confidence interval 0.74–0.93). Concentrations declined over time in horses undergoing treatment. NGAL is a novel biomarker that seems to have great potential for identifying septic synovitis and for monitoring the response to treatment of synovial infection in horses.