Acinetobacter johnsonii and Pseudomonas fluorescens are specific spoilage microorganisms (SSO) of aquatic products, and their metabolites are effective indicators for analyzing these two SSOs. However, the metabolome of a coculture of A. johnsonii and P. fluorescens is still unclear. The aim of this study was to investigate the metabolomic changes in A. johnsonii, P. fluorescens, and their co-culture by screening for metabolic markers. PCA and PLS-DA revealed that A, P, and AP groups were different from each other, indicating a significantly varying metabolic profile among them. Based on UHPLC information, 540 metabolites with significant differences, were identified in the ESI+ and ESI- modes, which covered 79 metabolic pathways. The different metabolites were mainly related to the pathways, such as taurine and hypotaurine metabolism:M, bile secretion:OS, and arginine biosynthesis:M were in the A vs AP group, while PPAR signaling pathway:OS, tropane, piperidine, and pyridine alkaloid biosynthesis:M, longevity regulating pathway-worm:OS, choline metabolism in cancer:HD, biosynthesis of phenylpropanoids:M, and amoebiasis:HD were in the P vs AP group. Therefore, this study may provide significant information regarding the metabolic mechanisms of A. johnsonii, P. fluorescens, and their co-cultures, which may provide insights on their role in deteriorating the quality of aquatic products. © 2020