The present study investigated the antioxidant and antimicrobial activities of naturally occurring Cystoseira barbata seaweed glyco-conjugates (CBGs), with a view to developing safer food preservatives. CBGs were successfully isolated, then chemically and structurally characterized. CBGs contained a high amount of polysaccharides (49.76%) that consisted mainly of neutral sugars (47.67%) and uronic acids (2.09%). The carbohydrate fraction was sulfated (13.81%) and conjugated with proteins (9.86%) and phenolic compounds (4.98%). Infrared spectroscopy of CBGs showed interactions between polyphenols, proteins and polysaccharides, which were characterized by α-type glycosidic bond and sulfate groups in the axial position of sugar residues. Neutral sugars analysis of CBGs by GC–MS revealed that conjugated polysaccharides were mainly composed of galactose (34.02%), fucose (26.25%) and mannitol (21.25%) with few amounts of other sugars such as glucose (5.78%), rhamnose (4.9%), xylose (3.22%) and mannose (2.22%). Analysis of the amino acid composition of CBGs showed a high level of essential amino acids (40.36%), in which threonine was the most relevant (10.28%). LC-QTOF-MS analysis of the phenolic fraction of CBGs showed a variety of phenolic compounds including flavonoids, phlorotannins and anthraquinone glycosides. CBGs exhibited potent antioxidant activities including radical scavenging activity, chelating ability and reducing power, and displayed noticeable antibacterial and antifungal activities, which may open the way to the development of a natural biopreservation strategy based on algae.