Temperate mesophotic reef ecosystems (TMREs) are among the least known marine habitats. Information on their diversity and ecology is geographically and temporally scarce, especially in highly productive large upwelling ecosystems. Lack of information remains an obstacle to understanding the importance of TMREs as habitats, biodiversity reservoirs and their connections with better-studied shallow reefs. Here, we use environmental DNA (eDNA) from water samples to characterize the community composition of TMREs on the central Chilean coast generating the first baseline for monitoring the biodiversity of these habitats. We analyzed samples from two depths (30 and 60m) over four seasons (spring, summer, autumn, and winter) and at two locations approximately 16 km apart. We used a panel of three metabarcodes, two that target all eukaryotes (18S rRNA and mitochondrial COI) and one specifically targeting fishes (16S rRNA). All panels combined encompassed eDNA assigned to 42 phyla, 90 classes, 237 orders, and 402 families. The highest family richness was found for the phyla Arthropoda, Bacillariophyta and Chordata. Overall, family richness was similar between depths but decreased during summer, a pattern consistent at both locations. Our results indicate that the structure (composition) of the mesophotic communities varied predominantly with seasons. We analyzed further the better-resolved fish assemblage and compared eDNA with other visual methods at the same locations and depths. We recovered eDNA from nineteen genera of fish, six of these have also been observed on towed underwater videos, while thirteen were unique to eDNA. We discuss the potential drivers of seasonal differences in community composition and richness. Our results suggest that eDNA can provide valuable insights for monitoring TMRE communities but highlight the necessity of completing reference DNA databases available for this region.