The cold-pressed oil from Japanese quince seeds (JQSO) possesses a favorable fatty acid composi-tion profile, a low oxidation rate, and contains antioxidants, sterols, and carotenoids, as reported in numerous publications addressing these chemical properties. This work is intended to add more information on physical characterization on molecular level on JQSO and the minor components present in the oil, using the differential scanning calorimetry (DSC), monolayer studies, and spec-troscopic methods—including UV-Vis absorption, total fluorescence, and FTIR. DSC was used to investigate the transitions during cooling and heating and to obtain thermodynamic parameters related to the melting and crystallization profiles of triglycerides (TGA) present in JQSO. Deconvo-lution of the melting profile indicated the presence of five main TGA structures. Langmuir mono-layer studies on JQSO revealed its high compressibility, indicating its good emulsification proper-ties. Meanwhile, viscoelastic modulus values suggest stronger intermolecular interactions, imply-ing that the oil monolayer is more resilient and can maintain its structural integrity under stress—a property typically associated with oils rich in saturated or monounsaturated fatty acids. Pres-ence of strong peaks in UV range of absorption spectrum indicates presence of phenolic acids, to-copherols, phenolic and carotenoid molecules. Multicomponent presence of bioactive molecules was investigated using Total Fluorescence Spectra which provide a comprehensive overview of all fluorescent molecules, with the most intense regions observed at 290 nm/330 nm and 360 nm/440 nm. The Total Synchronous Fluorescence spectra revealed two strong intensity areas with Δλ/excitation-emission pairs at 10–50 nm/300 nm and 40–140 nm/360 nm, indicating the presence of tocopherols, phenols, polyphenols, flavones, carotenoids, and their derivatives. No spectroscop-ic evidence on the chlorophyll presence in JQSO was found. ATR-FTIR spectrum confirmed the presence of groups characteristic for fatty acids and triacylglycerols, with special emphasizes on high degree of esterification of JQSO and indicating dominant presence of unsaturated fatty acids in this oil. The methods used provided the opportunity to perform a label-free, fast, and reliable determination of the properties of JQSO. Moreover, the presented data confirmed and visualized that crude, cold-pressed JQSO contains all the valuable ingredients reported elsewhere.