Our lack of fundamental knowledge about species occurrence, abundance, distribution, threats, and habitat requirements makes it challenging to conserve biodiversity in the face of rising human pressure. To obtain this information, practical and sensitive methods are required to identify and quantify the occurrence and diversity of rare, cryptic, and elusive species. Environmental DNA (eDNA) is a new technology that can help to discover and quantify biodiversity by addressing some of the limitations of conventional surveys. This study aimed to evaluate the biodiversity and composition of benthic species in the Lower Ciliwung River, Jakarta, Indonesia. The investigation was conducted from upstream to downstream of the river at the three sites during the dry season (July 2022). The DNA read sequences were used to examine the composition, relative abundance, and diversity of the samples. In this study, 1,492,975 initial reads were filtered down to 1,265,307 reads belonging to 27 species of benthic organisms. The taxa identified from the eDNA samples revealed that the most frequently detected species belonged to the family Siboglinidae, order Sabellida, and class Polychaeta. The composition of benthic species discovered in the eDNA water samples varied substantially (p ≤ 0.05). Osedax sp. (class Polychaeta) was the most prevalent species across all locations. The diversity index (H) ranged from low to moderate. eDNA offers potential advantages, but is hampered by DNA degradation, primer sensitivity, and the inability to distinguish between dead and living organisms.