Background and aims: The oxidative metabolism of polyunsaturated fatty acids (PUFAs) leads to bioactive isoprostanoids. The aim was to establish the associations of a complete urinary isoprostanoid profiling in a cohort study of carefully phenotyped obese subjects to determine possible potential differential implications for omega-6 PUFA- and omega-3 PUFA-derived isoprostanoids for obesity, metabolic indicators, and inflammation. Methods and results: PUFA peroxidation compounds were determined in urine samples from obese human subjects (n = 46) by liquid chromatography coupled to tandem mass spectrometry. Increased omega-6 arachidonic acid (AA) oxidation, mainly represented by 5-F2c isoprostane (5-F2c-IsoP) and metabolites of 15-F2t-IsoP, was associated with body mass index, glycated hemoglobin (HbA1c) and mean arterial blood pressure. In addition, we identified the omega-3 PUFA-derived urinary metabolites 14-F4t-NeuroP from docosahexaenoic acid (DHA) and 5-F3t-IsoP from eicosapentaenoic acid (EPA), which declined with age. The omega-3 to omega-6 oxidation ratio was a significant predictor of inflammation in obesity. Conclusion: The findings point to full urinary isoprostanoid profiling as a more sensitive measure of PUFA oxidative stress in obesity-induced metabolic complications compared with individual isoprostanoid measures. Furthermore, the results suggest the balance between the omega-3 and omega-6 PUFA oxidation as determinative for the consequences of oxidative stress on inflammation in obesity.