Understanding how microbial communities change with environmental degradation and restoration may offer new insights into the understudied ecology that connects humans, microbiota, and the natural world. Immunomodulatory microbial diversity and ‘Old Friends’ are thought to be supplemented from biodiverse natural environments, yet deficient in anthropogenically disturbed or degraded environments. However, few studies have compared the microbiomes of natural vs. human-altered environments and there is little knowledge of which microbial taxa are representative of ecological restoration—i.e. the assisted recovery of degraded ecosystems typically towards a more natural, biodiverse state. Here we use novel bootstrap-style resampling of site-level soil bacterial 16S rRNA gene environmental DNA data to identify genus-level indicators of restoration from a 10-year grassy eucalypt woodland restoration chronosequence at Mt Bold, South Australia. We found two key indicator groups emerged: ‘opportunistic taxa’ that decreased in relative abundance with restoration and more stable and specialist, ‘niche-adapted taxa’ that increased. We validated these results, finding seven of the top ten opportunists and eight of the top ten niche-adapted taxa displayed consistent differential abundance patterns between human-altered vs. natural samples elsewhere across Australia. Extending this, we propose a two-dimensional mapping for ecosystem condition based on the proportions of these divergent indicator groups. We also show that restoring a more biodiverse ecosystem at Mt Bold has increased the potentially immune-boosting environmental microbial diversity. Furthermore, environmental opportunists including the pathogen-containing genera Bacillus, Clostridium, Enterobacter, Legionella and Pseudomonas associated with disturbed ecosystems. Our approach is generalizable with potential to inform DNA-based methods for ecosystem assessment and help target environmental interventions that may promote microbiota-mediated human health gains.Craig Liddicoat, Philip Weinstein, Andrew Bissett, Nicholas J.C.Gelliea, Jacob G.Mills, Michelle Waycotta, Martin F.Bree