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Obesity is a multifactorial disease resulting in excessive accumulation of fat. Worldwide, obesity is an important public health problem, affecting a large proportion of the world population. The tender cactus Opuntia ficus-indica, commonly known in Mexico as “nopal”, is widely distributed in this country, Latin America, South Africa, and the Mediterranean area. Nopal cladodes are commonly marketed in different forms as fresh, frozen, or pre-cooked, and used as fresh green vegetable. The aim of this study was to evaluate the capability of nopal to improve the health condition of participants affected by obesity, in a physical and dietary intervention, through gut microbiota modification. These results were contrasted with the effect of nopal in the gut microbiota of normal weight participants. We describe the association among biochemical, anthropometric markers, and the gut microbiota diversity found in fecal samples of the obese and normal weight groups. The results presented in this work suggest that caloric restriction, addition of nopal to the diet and physical activity, promote changes in the gut microbiota in obese women, improving the host metabolism, as suggested by the correlation between some bacterial species with biochemical and anthropometrical parameters.