Communication provides the basis for social life. In ant colonies, the prevalence of local, often chemically mediated, interactions introduces strong links between communication networks and the spatial distribution of ants. It is, however, unknown how ants identify and maintain nest chambers with distinct functions. Here, we combine individual tracking, chemical analysis and machine learning to decipher the chemical signatures present on multiple nest surfaces. We present evidence for several distinct chemical 'road-signs' that guide the ants' movements within the dark nest. These chemical signatures can be used to classify nest chambers with different functional roles. Using behavioural manipulations, we demonstrate that at least three of these chemical signatures are functionally meaningful and allow ants from different task groups to identify their specific nest destinations, thus facilitating colony coordination and stabilization. The use of multiple chemicals that assist spatiotemporal guidance, segregation and pattern formation is abundant in multi-cellular organisms. Here, we provide a rare example for the use of these principles in the ant colony.