Flavonolignans occur typically in Silybum marianum (milk thistle) fruit extract, silymarin, which contains silybin, isosilybin, silychristin, silydianin, and their 2,3-dehydroderivatives, together with other minor flavonoids and a polymeric phenolic fraction. Biotransformation of individual silymarin components by human microbiota was studied ex vivo, using batch incubations inoculated by fecal slurry. Samples at selected time points were analyzed by ultrahigh-performance liquid chromatography equipped with mass spectrometry. The initial experiment using a concentration of 200 mg/L showed that flavonolignans are resistant to the metabolic action of intestinal microbiota. At the lower concentration of 10 mg/L, biotransformation of flavonolignans was much slower than that of taxifolin, which was completely degraded after 16 h. While silybin, isosilybin, and 2,3-dehydrosilybin underwent mostly demethylation, silychristin was predominantly reduced. Silydianin, 2,3-dehydrosilychristin and 2,3-dehydrosilydianin were reduced, as well, and decarbonylation and cysteine conjugation proceeded. No low-molecular-weight phenolic metabolites were detected for any of the compounds tested. Strong inter-individual differences in the biotransformation profile were observed among the four fecal-material donors. In conclusion, the flavonolignans, especially at higher (pharmacological) doses, are relatively resistant to biotransformation by gut microbiota, which, however, depends strongly on the individual structures of these isomeric compounds, but also on the stool donor.