Background: Due to the continuous global rise in obesity prevalence, foods rich in bioactive compounds are increasingly recognised for the management of several diseases. Objective: The present study aims to investigate whether an Opuntia stricta var. dillenii fruit peel extract, rich in betalains and phenolic compounds, is able to prevent obesity induced by a high-fat high-fructose diet in rats, along with the potential mechanisms of action underlying this effect. Results: The supplementation with Opuntia stricta var. dillenii extract obtained from the peel fruit partially prevents obesity development by attenuating HFHF-induced fat accumulation. This effect was observed predominantly in visceral adipose tissue, rather than in the subcutaneous depot. The obesity prevention was accompanied by the improvement of serum lipid profile. The mechanisms underlying the extract anti-obesity effect which were analysed in epididymal adipose tissue, involve preventing the rise in the availability of triglyceride synthesis substrates induced by high-fat high-fructose feeding, the inhibition of triglyceride assembly, and in the case of the high dose, increased lipolysis. Conclusions: According to these results, the peel wastes of Opuntia stricta var. dillenii fruit represent a promising natural source of bioactive compounds for obesity prevention. Nevertheless, these preclinical effects should be replicated in further studies in human beings.