The fruit extract of Opuntia stricta var. dillenii (OSDE) has been recognized for its effects on hepatic steatosis, but the compounds responsible for this activity have yet to be precisely identified. This work aimed to evaluate the anti-steatotic effect of OSDE and its different fractions obtained by fast centrifugal-partition chromatography (FCPC) to identify the compounds potentially responsible for this biological activity. Hepatic lipid accumulation and triglyceride content were evaluated, as well as cellular antioxidant activity and inhibition of lipid peroxidation. Pool 1-Pool 4 showed lower lipid accumulation than OSDE in liver cells, while a greater reduction in triglyceride levels, even lower than OSDE and lovastatin (LOV), was observed for Pool 1, 9, and 10. Compared to OSDE, Pools 1,6, 7, and 12 showed higher cellular antioxidant effects, whereas OSDE showed better lipid peroxidation inhibition than all of Pools. Quinic and piscidic acids were the main bioactive present in Pool 1, exhibiting +1597 % and + 997 % increases in their content related to OSDE, respectively. Likewise, the most abundant compounds in Pool 2- Pool 4 were betalains such as betanin and isobetanin, with +163 % and + 162 % of increases in their concentration related to OSDE, respectively. Antioxidant effects in Pools 6 and 7 correlated with higher phenolic acid concentration. OSDE significantly reduced triglyceride levels in a steatotic-induced model. Although OSDE showed anti-steatotic effects, these effects were more pronounced for some of its constituents in FCPC Pools. Results suggested that these compounds might be potentially responsible for this anti-steatotic effect. FCPC fractionation facilitated the separate biological evaluation of OSDE constituents and thus identified them. Future studies should focus on validating these anti-steatotic effects in in vivo models.