There is a complex interplay of criminal groups’ control over land, illicit activities, and forest cover change in the Colombian Andes-Amazon region. This area is dealing with diverse forms of conflict and peace, directly impacting landscape connectivity. While many studies have documented rapid deforestation after the peace agreement in 2016, we know little about the effect of these socio-political changes on the state of landscape connectivity. We disentangle habitat from connected habitat in forest ecosystems using the Landsat archive and landscape connectivity indices. We find that in the Andes-Amazon region during 2000–2020, connected habitat loss reached 18%, while habitat loss was 13%. This result is worrisome because it indicates that well-connected patches are more fragmented and isolated, affecting the natural connections between the Andes and Amazon biogeographical regions and the movement ability of species. The Colombian government should conduct a strategic peacebuilding process incorporating structural changes that prevent the increase of large-scale extractive activities that are often illegal in the region. While finding a balance between extractive activities and conservation remains a big challenge, legal land tenure, census/taxation, and specific agreements with local actors can initially prevent deforestation. We discourage localized military actions and the return of aerial fumigation of coca fields, which rather than stop deforestation might exacerbate land cover change deeper into pristine forests. • De-escalation of conflict coincides with intra-annual deforestation dynamics in Colombia. • Connected habitat loss (18%) is higher than habitat available (13%) during 2000–2020. • Rapid fragmentation of well-connected forest patches can affect the biological connection between the Andes-Amazon biomes. • Reachable habitat for species is now less than the habitat available. • Conservation strategies should focus on developing agreements with local communities.