The effects of arbuscular mycorrhizal fungi (AMF) inoculation on the chemical composition of the fruits and cladodes of two Opuntia ficus-indica cultivars, characterized by their red and yellow fruit color, were investigated under field conditions. AMF treatment was found to significantly influence the concentration of phytonutrients in the fruits. The concentrations of betacyanin and betaxanthin increased by 1.2- and 1.9-fold in red and yellow fruits, respectively. The polyphenol content increased by 50%, with piscidic acid being the most abundant polyphenol in the red fruits. A similar increase in ascorbic acid was observed in the yellow fruits. Regarding the cladodes, AMF treatment was found to significantly affect macronutrient levels, with glucose and fructose contents being 90% and 34% higher, respectively. Additionally, cladodes from plants grown with AMF inoculation showed a 20% increase in ascorbic acid and phosphorus. These results demonstrate cultivar- and part-of-plant-dependent effects of AMF inoculation and confirm the nutritional and sustainable potential of Opuntia ficus-indica, particularly when coupled with mycorrhizal biofertilization practices.