Display omitted •The effects of freeze-dried O. dillenii cladodes (FDOd) on gut microbiota were reported.•FDOd has bioactive compounds with prebiotic and antioxidant properties.•FDOd induced positive changes in intestinal microbiota composition.•FDOd induced the production of several metabolites during colonic fermentation.•FDOd kept antioxidant capacity during colonic fermentation. This study evaluated the physicochemical properties, antioxidant capacity, and potential prebiotic effects on the human intestinal microbiota of freeze-dried Opuntia dillenii (Ker Gawl). Haw (Cactaceae) cladodes (FDOd). FDOd had high levels of total fiber (52.36 g/100 g), especially soluble fiber (33.36 g/100 g), as well as minerals, such as potassium (4415.49 mg/100 g), calcium (1593.25 mg/100 g), magnesium (853.55 mg/100 g), and phosphorus (209.04 mg/100 g), high total chlorophyll content (912.00 mg/100 g), presence of various phenolic compounds, such as catechin (41.00 mg/100 g), myricetin (785.00 mg/100 g), isorhamnetin (5.00 mg/100 g), rutin (42 mg/100 g), and antioxidant activity (ABTS•+: 0.51 µmol TEAC/g; DPPH•: 0.32 µmol TEAC/g; FRAP: 2.75 µmol FeSO4/g). Fermentation of FDOd using human fecal inoculum changed the composition and metabolic activity of intestinal microbiota, increasing the relative abundance of Ligilactobacillus (0.03 %–16.44 %), Lactiplantibacillus (0.00 %–1.11 %), and Agathobacter (1.80 %–4.23 %), and decreasing the relative abundance of Prevotella_9 (25.08 %–0.15 %) and Succinivibrio (24.77 %–1.54 %). FDOd influenced the metabolic profile of the intestinal microbiota with the production of various metabolites, including short- and medium-chain fatty acids, organic acids, essential amino acids, and other compound classes with health-promoting properties. The results indicate FDOd as a new ingredient with prebiotic properties in the human intestinal microbiota.