The Critically Endangered western swamp turtle (Pseudemydura umbrina), a long-lived species endemic to southwest Australia, has experienced extensive habitat loss and fragmentation, and its remaining seasonal wetland habitat is threatened by a drying climate. Assisted colonisation trials south of the turtle’s indigenous range commenced in 2016 and research is ongoing. When intentionally translocating species, it is essential to evaluate metrics associated with the translocated population (e.g., dispersal, establishment and impacts to the recipient ecosystem). We developed environmental DNA (eDNA) methods to 1) detect P. umbrina, and 2) determine their diet at assisted colonisation sites. Effective monitoring protocols for conservation translocation require techniques that account for the dispersal and cryptic nature of translocated animals, such as non-invasive molecular eDNA approaches. First, we successfully designed, validated, and optimised a sensitive P. umbrina-specific eDNA assay. We detected P. umbrina at the assisted colonisation wetland in 42% (7 / 17) of eDNA sample sites, and the probability of a positive eDNA detection was significantly associated with the distance from P. umbrina, reflecting spatial distribution of the target species. Second, to assess the food web and ecosystem impacts of P. umbrina in a novel environment, we applied DNA metabarcoding to analyse their diet using universal mitochondrial primers targeting their macroinvertebrate and tadpole prey. DNA sequences obtained from faecal samples and cloacal swabs revealed a more precise diet characterisation when compared to the more-invasive method of stomach flushing. As demonstrated here, non-invasive molecular eDNA approaches be effectively implemented to evaluate assisted colonisation performance metrics.