The nutritional and health properties of algae make them perfect functional ingredients for nutraceutical and cosmeceutical applications. In this study, thePhaeodactylum tricornutumBohlin (Phaeodactylaceae), a pleiomorphic diatom commonly found in marine ecosystems, was investigated. The in vitro culture conditions used favoured the fusiform morphotype, characterized by a high accumulation of neutral lipids, as detected by fluorescence microscopy after BODIPY staining. These data were confirmed by HPLC-DAD-APCI-MS/MS analyses carried out on the ethanolic extract (PTE), which showed a high content of xanthophylls (98.99%), and in particular of fucoxanthin (Fx, 6.67 g/100 g PTE). The antioxidant activity (ORAC, FRAP, TEAC and β-carotene bleaching) and photostability of PTE and Fx against UVA and UVB rays were firstly evaluated by in vitro cell-free assays. After this, phototoxicity and photoprotective studies were carried out on in vitro reconstructed human epidermidis models. Results demonstrated that PTE (0.1% Fx) and 0.1% Fx, both photostable, significantly (p< 0.05) reduce oxidative and inflammatory stress markers (ROS, NO and IL-1α), as well as cytotoxicity and sunburn cells induced by UVA and UVB doses simulating the solar radiation, with an excellent safety profile. However, PTE proved to be more effective than Fx, suggesting its effective and safe use in broad-spectrum sunscreens.