Hemp (Cannabis sativa L.) is gaining interest as a food ingredient due to the nutritional value of its seeds, particularly their high content of polyunsaturated fatty acids (PUFAs), including linoleic (LA, 18:2n-6) and alpha-linolenic (ALA, 18:3n-3) acids. These essential fatty acids are associated with health-promoting effects, yet their bioaccessibility after digestion is crucial to exert such benefits. This study evaluated the fatty acid bioaccessibility, total phenolic content, and antioxidant capacity of five hemp seed varieties ('Futura 75', 'Futura 83', 'Earlina 08FC', 'Felina 32', and 'Henola') after in vitro digestion. LA and ALA represented up to 70% of total lipids, with 'Earlina 08FC' showing the highest content. However, PUFAs exhibited lower bioaccessibility than saturated fatty acids due to oxidative degradation. Eicosadienoic and stearidonic acids, though less abundant, showed higher bioaccessibility in 'Futura 75' and 'Henola', respectively. Finally, the former whole seed resulted in lower total phenolic content compared with the other varieties studied, but after in vitro digestion it also showed significantly higher bioaccessibility of phenols and higher antioxidant capacity. These findings suggest that targeted breeding and processing could enhance PUFA bioaccessibility, with some varieties showing potential for nutraceutical applications.