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Phaeodactylum tricornutum (P. triconutum) gained high interest for use in human nutrition. We aimed to investigate the effect of photoautotrophically cultivated Phaeodactylum tricornutum on genotoxicity, cytotoxicity, formation of reactive oxygen species and apoptosis in Caco-2 and HT-29 cells. Furthermore, the effect of processing (sonication, ball-milling) on these parameters was assessed.P. triconutum dose-dependently induced DNA strand breaks (2 µg dry matter/mL – 200 µg dry matter/mL) in both cell lines. These breaks can mostly be repaired after a recovery time of 2 h. Using processing methods did not affect the genotoxicity. We found that a lipophilic fraction, but not fucoxanthin, might be responsible for the DNA damage. Neither the P. triconutum preparations nor fucoxanthin affected cell proliferation or revealed an apoptotic or necrotic activity.In conclusion, P. triconutum showed a genotoxic potential in intestinal cells, mainly mediated by lipophilic constituents. Sonication and ball-milling might be considered as safe methods for processing of P. triconutum.