AbstractMarine algae are one of the most important sources of high‐value compounds such as polar lipids, omega‐3 fatty acids, photosynthetic pigments, or secondary metabolites with interesting features for different niche markets. Acetabularia acetabulum is a macroscopic green single‐celled alga, with a single nucleus hosted in the rhizoid. This alga is one of the most studied dasycladalean species and represents an important model system in cell biology studies. However, its lipidome and pigment profile have been overlooked. Total lipid extracts were analyzed using hydrophilic interaction liquid chromatography‐high resolution mass spectrometry (HILIC‐HRMS), tandem mass spectrometry (MS/MS), and high‐performance liquid chromatography (HPLC). The antioxidant capacity of lipid extracts was tested using DPPH and ABTS assays. Lipidomics identified 16 polar lipid classes, corresponding to glycolipids, betaine lipids, phospholipids, and sphingolipids, with a total of 191 lipid species, some of them recognized by their bioactivities. The most abundant polar lipids were glycolipids. Lipid classes less studied in algae were identified, such as diacylglyceryl‐carboxyhydroxymethylcholine (DGCC) or hexosylceramide (HexCer). The pigment profile of A. acetabulum comprised carotenoids (17.19%), namely cis‐neoxanthin, violaxanthin, lutein and β,β‐carotene, and chlorophylls a and b (82.81%). A. acetabulum lipid extracts showed high antioxidant activity promoting a 50% inhibition (IC50) with concentrations of 57.91 ± 1.20 μg · mL−1 (438.18 ± 8.95 μmol Trolox · g−1 lipid) in DPPH and 20.55 ± 0.60 μg · mL−1 in ABTS assays (918.56 ± 27.55 μmol Trolox · g−1 lipid). This study demonstrates the potential of A. acetabulum as a source of natural bioactive molecules and antioxidant compounds.