This study aimed to investigate the bioaccessibility and biostability of carotenoids, vitamin E isomers, and individual polyphenolic compounds after the in vitro gastrointestinal digestion of two types of berries (raspberry and blackberry fruits). The results of the polyphenols analysis showed that raspberry fruits contained higher concentrations of hydroxybenzoic acids, hydroxycinnamic acids, flavanols, and flavonols compared to blackberry fruits, but exhibited the lowest bioaccessibility values for all the studied polyphenol classes. Ellagic acid represented 13.63% and 2.65% of the hydroxybenzoic acids in raspberry and blackberry fruits. The hydroxybenzoic acids exhibited the highest bioaccessibility index in the intestinal phase of both types of berries, and gallic acid emerged as one of the most bioaccessible phenolic compounds. The bioaccessibility of carotenoids ranged between 15.7 and 17.30% for lutein, 5.52 and 7.56% for astaxanthin, and 7.85 and 9.93% for canthaxanthin, with elevated values being observed in raspberry fruits. Although vitamin E and carotenoids follow a similar path for absorption, the bioaccessibility of vitamin E isomers was higher than that of carotenoids, with γ-tocopherol being the most bioaccessible isomer in both raspberries and blackberries. Knowing the bioaccessibility of food constituents during digestion is crucial, as the potential effectiveness of bioactives for human health largely depends on the bioavailability of these molecules.