The microalgae Phaeodactylum tricornutum (PT) contains valuable nutrients such as proteins, polyunsaturated omega-3 fatty acids (n-3 PUFA), particularly eicosapentaenoic acid (EPA) and some docosahexaenoic acid (DHA), carotenoids such as fucoxanthin (FX), and beta-glucans, which may confer health benefits. In a randomized intervention trial involving 22 healthy individuals, we administered for two weeks in a crossover manner the whole biomass of PT (5.3 g/day), or fish oil (FO) containing equal amounts of EPA and DHA (together 300 mg/day). In an additional experiment, sea fish at 185 g/week resulting in a similar EPA and DHA intake was administered in nine individuals. We determined the bioavailability of fatty acids and carotenoids and assessed safety parameters. The intake of PT resulted in a similar increase in the n-3 PUFA and EPA content and a decrease in the PUFA n-6:n-3 ratio in plasma. PT intake caused an uptake of FX that is metabolized to fucoxanthinol (FXOH) and amarouciaxanthin A (AxA). No relevant adverse effects occurred following PT consumption. The study shows that PT is a safe and effective source of EPA and FX—and likely other nutrients—and therefore should be considered as a future sustainable food item.