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The physicochemical characterization of fresh, undiluted, cold-pressed oil from elderberry seeds (EO) is presented. The results showed EO's uniqueness for the 93 % presence of essential fatty acids, including linoleic n-6 (41 %), α-linolenic n-3 (38 %), and oleic n-9 (13 %) acids with favorable ratios for human nutrition, n-3/n-6 = 0.93. A γ-tocopherol is the dominant tocopherol (96 %), with a concentration of 20.62 mg/100 g, indicating low oil oxidative stability. DSC heating and cooling traces determined the thermal properties. These results also revealed the presence of metastable triacylglycerol (TAG) structures composed of polyunsaturated fatty acids. The presence of characteristic groups for fatty acids and TAGs in EO was confirmed by FTIR-ATR spectra. For the first time, Langmuir monolayer studies on EO revealed its low compressibility, indicating its low emulsifiability, and the presence of minor components of EO, including tocopherols, phenolic acids, polyphenols, flavonoids, and carotenoids, was determined using UV-Vis absorption and fluorescence excitation-emission matrix (EEM) along with the chemometric method., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)