In patients with a mild to moderate bleeding disorder (MBD) and abnormal light transmission aggregometry (LTA), a platelet function defect (PFD) is suspected. However, in many patients with PFD, the underlying mechanism remains elusive. Given the essential role of lipids in platelet signaling, platelet lipid profiles in MBD patients with unexplained PFD may provide valuable diagnostic and mechanistic insights. This prospective cohort study investigated platelet lipidomes in patients with PFD of unknown cause from the Vienna Bleeding Biobank (VIBB). Using a standardized lipidomics workflow, we analyzed platelets from 27 patients and 19 age- and sex-matched controls and found that sex-specific lipid shifts emerged exclusively within the patient cohort, with greater deviations in females. Furthermore, lipid alterations correlated with impaired platelet aggregation and were predictive of responses to ADP and TRAP-6 stimuli in LTA experiments. Baseline and stimulated platelet analyses in a female subgroup showed intrinsic lipidomic changes, including upregulated polyunsaturated triacylglycerols (PUFA-TG), acylcarnitines (CAR), and reduced lysophosphatidylethanolamines (LPE). This study emphasizes lipidomic profiling as a promising diagnostic tool for unexplained platelet dysfunction and highlights TG, CAR, and LPE as potential therapeutic targets. Further research into lipid-driven platelet regulation may advance personalized treatments and improve clinical outcomes for patients with MBD.