Non-invasive sampling of biological materials, such as faeces, provides valuable ecological and evolutionary information for elusive, threatened, or ancient species. In arid regions, rodent middens, which are accumulations of faeces, plant remains, bones, and insects, can persist for millennia, preserving exceptional palaeoecological records. Yet, identifying the species responsible for their construction remains challenging, as morphological traits are often ambiguous or degraded. Here, we report the first application of High-Resolution Melting (HRM) analysis to DNA extracted from modern and ancient rodent midden faeces from the Atacama Desert. By integrating faecal morphology with a qPCR-HRM assay targeting a 16S rDNA fragment, followed by Sanger sequencing, we distinguished two faecal morphotypes corresponding to Octodontidae and Cricetidae. HRM successfully amplified 15 samples, including two dated to 2,410 and 2,350 cal BP, generating distinct melting profiles. Sequencing comparisons of representative samples further supported the attribution of these profiles to Octodon degus and Phyllotis spp. respectively, consistent with morphological assignments of faeces and ecological expectations. Nine samples failed amplification, highlighting the importance of sample preservation. Our results show that HRM, integrated with morphological assessment, represents a promising, rapid, cost-effective, and non-invasive approach for the taxonomic assessment of faecal and paleofecal material, offering new opportunities to link molecular and environmental data in palaeoecological studies.