Carnivores represent important ecological components as apex predators, but their diversity is still poorly described in Saudi Arabia due to a deficiency of field data. This study provides the first molecular assessment of carnivores across southern Saudi Arabia using a multilocus DNA barcoding approach (COI, Cytb, and 16S). Out of 155 biological samples (scats and tissues), we successfully generated and analyzed 152 sequences, identifying 11 species (Panthera pardus, Caracal caracal, Acinonyx jubatus, Felis lybica, Vulpes vulpes, Canis lupus, Canis lupus familiaris, Hyaena hyaena, Genetta genetta, Ichneumia albicauda, and Herpetes javanicus). Phylogenetic and haplotype network analyses uncovered clear resolution at the species level with substantial intraspecific variation, especially within Vulpes vulpes, Canis lupus, and Panthera pardus. We have also confirmed, for the first time since the sighting in 2014, the presence of the critically endangered Arabian leopard, Panthera p. nimr. The observed high genetic diversity among the studied leopard samples likely reflects the temporal divergence of the sampled specimens rather than the true current population diversity. The cheetah (Acinonyx jubatus) samples in our study likely represent introduced individuals from Africa via illegal trade, rather than native Arabian populations. Genetic divergence among haplotypes reveals historical isolation and recent population expansions favored by habitat fragmentation. The proposed methodology of integrating molecular barcoding with traditional field approaches may hold great potential for the protection of the elusive and threatened carnivore fauna of Saudi Arabia.