Occurring across all southern hemisphere continents except Antarctica, old, climatically buffered, infertile landscapes (OCBILs) are centres of biological richness, often in biodiversity hotspots. Among a matrix of young, often disturbed, fertile landscapes (YODFELs), OCBILs are centres of endemism and diversity in the exceptionally rich flora of the south-west Australian global biodiversity hotspot, home to Noongar peoples for ≥ 48 000 years. We analysed contemporary traditional Noongar knowledge of adjacent OCBILs (e.g. granite outcrops) and YODFELs (e.g. creekline fringes) both at a single site and in two larger areas to test whether patterns of disturbance dictated by Noongar custom align with OCBIL theory. We found that Noongar traditional knowledge reflects a regime of concentrated YODFEL rather than OCBIL disturbance—a pattern which aligns with maximal biodiversity preservation. SIMPER testing found traditional Noongar OCBIL and YODFEL activities are 64–75% dissimilar, whereas Pearson’s chi-square tests revealed camping, burning, travelling through country and hunting as primarily YODFEL rather than OCBIL activities. We found that Noongar activities usually avoid OCBIL disturbance. This combined with high floristic diversity following enduring First Peoples’ presence, suggests that traditional Noongar knowledge is valuable and necessary for south-west Australian biodiversity conservation. Similar cultural investigations in other OCBIL-dominated global biodiversity hotspots may prove profitable.