Osteoarthritis (OA), osteochondrosis (OC), and synovial sepsis in horses cause loss of function and pain. Reliable biomarkers are required to achieve accurate and rapid diagnosis, with synovial fluid (SF) holding a unique source of biochemical information. Nuclear magnetic resonance (NMR) spectroscopy allows global metabolite analysis of a small volume of SF, with minimal sample preprocessing using a noninvasive and nondestructive method. Equine SF metabolic profiles from both nonseptic joints (OA and OC) and septic joints were analyzed using 1D 1H NMR spectroscopy. Univariate and multivariate statistical analyses were used to identify differential metabolite abundance between groups. Metabolites were annotated via 1H NMR using 1D NMR identification software Chenomx, with identities confirmed using 1D 1H and 2D 1H 13C NMR. Multivariate analysis identified separation between septic and nonseptic groups. Acetate, alanine, citrate, creatine phosphate, creatinine, glucose, glutamate, glutamine, glycine, phenylalanine, pyruvate, and valine were higher in the nonseptic group, while glycylproline was higher in sepsis. Multivariate separation was primarily driven by glucose; however, partial-least-squares discriminant analysis plots with glucose excluded demonstrated the remaining metabolites were still able to discriminate the groups. This study demonstrates that a panel of synovial metabolites can distinguish between septic and nonseptic equine SF, with glucose the principal discriminator.