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[Display omitted] • Ulva rigida and Gracilaria fisheri improve gut health in vitro differently. • Seaweed polysaccharides are not digestible by small intestinal enzymes. • Seaweeds and polysaccharides stimulate beneficial microbes and SCFAs output in vitro. • The red seaweed Gracilaria fisheri has more potential as a source of prebiotics. This study explored the gut health potential of the green seaweed Ulva rigida (SW-U) and the red seaweed Gracilaria fisheri (SW-G), as well as polysaccharide-enriched extracts (PF-U and PF-G, respectively). The polysaccharide-enriched extracts were not digestible by small intestinal enzymes, but the morphology of samples was changed. After 24 h in vitro fermentation, seaweeds and polysaccharide-enriched extracts significantly increased (p < 0.05) production of total short-chain fatty acids (SCFAs) (29.4–35.4 µmoL/mL) compared to the control (17.9 µmoL/mL). The G. fisheri showed more potential for improving gut health than U. rigida. Particularly, PF-G induced butyric acid production comparable to that of inulin control (5.4 and 6.9 µmoL/mL) and the highest production when compared with other substrates (1.8–3.3 µmoL/mL). While SW-G stimulated the growth of beneficial bacteria, including Roseburia and Faecalibacterium. These findings further demonstrate that seaweeds and their derived polysaccharides have the potential to be used as dietary supplements with gut health benefits. [ABSTRACT FROM AUTHOR]