Longan seeds account for 17 wt.% of the fruit weight, but most longan seeds are discarded during longan processing or fresh consumption, and comprehensive utilization is not achieved. To clarify the compositional characteristics and biological activities of longan seed oils from different varieties, develop and utilize longan seed oils, and improve their utilization rate, longan seeds of eight varieties, namely Shixia, Chuliang, Daguangyan, Shuizhang, Fenglisui, Shuangzimu, Fuyan and Thai longan, are selected, and their oils are extracted and analyzed. The fatty acid compositions of longan seed oils from different varieties are analyzed by gas chromatography. Structural lipids including triglycerides, diglycerides and phospholipids in the oils are separated by thin-layer chromatography, and fatty acid compositions of each structural lipid are further determined. Meanwhile, DPPH radical scavenging capacity and antibacterial activity of the oils are detected, and the correlation between fatty acid compositions and biological activities is analyzed. The results show that six saturated fatty acids and five unsaturated fatty acids are detected in all eight longan seed oils, with the unsaturated fatty acid proportion ranging from 55.74 wt.% to 81.44 wt.%. Fuyan, Chuliang and Daguangyan exhibit outstanding advantages in total fatty acid content and unsaturated fatty acid content. Among structural lipids, triglycerides possess the most abundant fatty acid species, and the overall content is ranked as Shuizhang > Fuyan > other varieties. The antioxidant activity and inhibitory activity against Staphylococcus aureus of Shuizhang longan oil are the optimal, which are 1.88 times and 63.67 times those of other varieties, respectively. Correlation analysis indicates that fatty acids (C16:0, C18:1-9, C18:2, C18:3n-3) are significantly positively correlated with antioxidant activity. It is demonstrated that longan seed oils of different varieties are rich in unsaturated fatty acids. Fuyan, Chuliang and Daguangyan are superior varieties in fatty acid composition, and Shuizhang longan oil shows the best performance in antioxidant and antibacterial activities. Fatty acids such as C16:0, C18:1-9, C18:2 and C18:3n-3 are important contributors to their biological activities. The findings provide a theoretical basis for the functional development and utilization of longan seed oils and the screening of excellent varieties.