Antarctica is commonly regarded as the most pristine place on Earth and is safeguarded by the Antarctic Treaty System (ATS). The ATS designates the continent as a “natural reserve, devoted to peace and science” and sets out environmental principles regulating human activities. The obligations under the ATS include providing environmental impact assessments and leaving the environment minimally impacted after all operations. The associated human footprint inevitably increases as human activity increases in intensity and diversity on the continent. The human footprint is not clearly defined, and most investigation has focused on visible disturbances of physical (e.g., soil disturbance) and chemical (e.g., hydrocarbon spills) nature. The unseen human footprint remains to be investigated and quantified, which includes released genetic materials and human-associated microorganisms. Given the extremely low biomass in most of terrestrial Antarctica, these materials' presence can be a reliable indicator of human presence and activities. As part of New Zealand’s effort to carry out an evidence-based risk assessment of the McMurdo Dry Valley ecosystem (DryVER project), this research quantified human impact, in the form of DNA released from humans and human-associated microbiota, across spatial and temporal scales. This project was part of the MBIE grant entitled “Evidence-based Risk Assessment of the McMurdo Dry Valley Ecosystem”. Areas of human activity, including seasonal field camps and facilities zones, were selected as sampling sites in the McMurdo Dry Valleys (MDVs), from which soil samples were collected. Human-specific genetic and microbial markers were used with digital PCR to quantify human presence at the lowest possible detection limit across spatial scales. A marker targeting a human MT-CYB gene region was the best marker for detecting and predicting human trace up to 1 km from a highly occupied site. Human impact derived from mtDNA was accumulative with occupancy and greatest closest to a campsite. A marker targeting 16S rRNA of Bacteroides dorei, a human-associated gut bacterium, was used to detect faecal trace. The faecal signal was generally localised to the toilet area of a camp and was too low to predict the distance of detectable signal, regardless of the intensity of occupancy. A DNA longevity experiment was established in situ in Miers Valley to investigate DNA persistence in a cold desert across temporal scales. The results from this experiment demonstrated that intracellular and extracellular DNA remained detectable with endpoint PCR for at least two years. DNA persistence was not significantly affected by soil sterility or UV exposure but did accumulate damage over time. The direct outcome of this research will be a review of the environmental management systems in place, namely the McMurdo Dry Valleys Antarctic Specially Managed Area (ASMA) Management Plan. Better management will safeguard the unique MDV ecosystems by using the best practice to minimise anthropogenic impact and strengthen New Zealand’s stewardship over the Ross Dependency as one of the original 12 signatories to the Antarctic Treaty.