This study presents an extensive compositional characterization of biodiesel produced from sweet almond (Prunus amygdalus “dulcis”) seed oil, complementing earlier analyses of its physicochemical and fuel performance properties. While previous investigations evaluated parameters such as viscosity, density, flash point, acid value, and cold-flow behavior, the present work focuses on the detailed molecular composition of both the feedstock and its resulting biodiesel, with particular emphasis on the fatty acid (FA) and fatty acid methyl ester (FAME) profiles. Comprehensive knowledge of these molecular constituents is critical for predicting biodiesel stability, combustion efficiency, and compliance with established international standards. The biodiesel was synthesized via base-catalyzed transesterification using a heterogeneous calcium oxide (CaO) catalyst derived from locally available materials. Gas Chromatography–Mass Spectrometry (GC/MS) was used to identify and quantify the dominant fatty acids in the crude almond oil and the corresponding methyl esters formed after conversion. GC/MS analysis of the raw oil revealed a rich fatty acid composition dominated by oleic acid (20.78%), n-hexadecanoic acid (10.65%), and octadecadienoic acid (8.47%). The distribution reflects an oil rich in both monounsaturated and polyunsaturated fatty acids, a desirable characteristic for biodiesel production due to its potential to enhance key fuel properties. Following transesterification, these fatty acids were transformed into their respective methyl esters, with oleic acid methyl ester (20.91%) and octadecadienoic acid methyl ester (18.32%) identified as the most abundant FAMEs. The predominance of these unsaturated esters strongly correlates with the biodiesel’s previously reported superior cold-flow characteristics, including a pour point of -9 °C and a cloud point of -3 °C. High monounsaturated ester content improves fluidity and contributes to oxidative stability, while polyunsaturated esters enhance low-temperature operability. This correlation underscores the significance of molecular-level profiling in assessing and predicting biodiesel performance. Overall, the findings confirm that the fatty acid composition of almond seed oil plays a crucial role in determining the quality of the resulting biodiesel and its ability to meet ASTM D6751 and EN 14214 specifications. The results validate sweet almond seed oil as a viable, sustainable, and technically promising non-conventional feedstock for biodiesel production in Nigeria. Its adoption could support cleaner energy alternatives, improve energy security, and contribute meaningfully to addressing the food–fuel dilemma.