Display omitted •RMF enzyme may be used to extract selectively IG5 or in combination with IG2.•Viscozyme enzyme selectively extracted triglycosides IG1 and IG2.•IG5 was the most potent inhibitor of NO2− reducing its production by 71.6±4.8%.•Enzymatic SC-CO2 released the glycosides with higher anti-inflammatory activity.•The optimum conditions to obtain selectivelyIG5 were 40°C, pH4 and 10% ethanol. Isorhamnetin conjugates from Opuntia ficus-indica (L.) Mill (nopal) were obtained using enzymatic hydrolysis pretreatment under supercritical carbon dioxide (SC-CO2) conditions. Factorial designs and discriminate analysis were used to evaluate the influence of pressure, temperature, pH, time and aqueous ethanol solution on the total extraction yield and the isorhamnetin recovery using the enzymes Rapidase Maxi Fruit (RMF) and Viscozyme. Isorhamnetin triglycosides isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside (IG1) and isorhamnetin-3-O-glucosyl-rhamnosyl-pentoside (IG2) and a diglycoside isorhamnetin-3-O-glucosyl-rhmanoside (IG5) were found in the extracts. The isorhamnetin profile pattern was dependent on the enzyme and process conditions used. The extract containing higher amounts of IG5 was the most potent inhibitor of nitric oxide reducing its production by 71.6±4.8%. Selective extraction of IG5 was achieved using RMF enzyme and optimal conditions determined by a response surface methodology. The use of enzyme under SC-CO2 conditions affected the release of isorhamnetin conjugate profiles which induced remarkable differences in the anti-inflammatory activity.