Totoaba macdonaldi is an endangered fish species endemic from the Gulf of California, and was a source of livelihood for people along the coast. Due to overexploitation, the Mexican government banned the fishery in 1975. However, it is still illegally fished and exported to China because the gas bladder is highly valued in traditional Chinese medicine. Despite its status, little is known about Totoaba biology. In order to optimize Totoaba farming, and to include the knowledge about its trophic ecology in conservation efforts, the diet of this fish has to be well characterized. Therefore, the aim of this study was to standardize a metabarcoding protocol to describe the diet by next-generation sequencing. We dissected four wild Totoaba individuals that had been seized by Mexican law enforcement agents, and we collected the stomach contents. From extracted DNA, we generated four representative amplicon libraries for cephalopods, chordates, marine invertebrates, and eukaryotes. After sequencing, we identified 12 different prey indicating Totoaba’s preference for fish (9 species), mainly Pacific anchovy (Cetengraulis mysticetus) and Flathead grey mullet (Mugil cephalus). Other identified prey were members of the Euphausiidae family (krill). Compared with previous studies based on morphological recognition of prey in 35 stomachs, we identified more taxa and species (18 and 14 vs 15 and 3, respectively) in only 4 stomachs. Moreover, we found seven new prey species. Our work confirms that metabarcoding is an effective method to study the feeding habits of this species, providing the tools to further analyze Totoaba diet.