In this research we analyzed land cover/use processes and their impact on biodiversity in the Megalopolis of Mexico City. We used land cover/use databases from 1976 and 2018, both validated, improved and adapted for conducting landscape dynamic analysis. We also included records of 159 threatened species of fungi, vascular plants and vertebrates to construct spatially explicit biodiversity richness models based upon niche ecological algorithms. The results showed that human settlement encroachment was the main factor driving land cover/use changes, significantly affecting rural and natural landscapes. The extent and location of the dramatic shrinking of agricultural lands was clearly demonstrated. Afforestation was the second most important land cover/use process occurring mainly on native grasslands and shrublands. Biodiversity richness was depleted substantially, affecting about 35 % of the most important biodiversity hot spots and rendering the remainder more vulnerable due to extensive fragmentation of native ecosystems. The results are discussed in the light of the implications of the value of interdisciplinary methodological approaches, potential water recharge, governance of territorial disputes, loss of cultural heritage and poorly implemented environmental policies. Furthermore, the study highlights the urgent need to generate an innovative model for development which gives equal importance to the conservation of natural and rural landscapes as a fundamental form of subsistence for human settlements. Protecting biocultural heritage is of paramount importance. The region's genetic resources and cultural diversity are unique and have played a fundamental role in providing various benefits from nature to urban and rural inhabitants. These findings can serve as a guide for other similar megacities around the world.