Pseudomonas aeruginosa biofilms expand in interstitial spaces by twitching motility, forming an intricate network of interconnected trails along which cells preferentially migrate. However, the factors involved in coordinating such self-organizing behaviour were unknown. Here, Gloag et al. show that cells migrating outwards from the biofilm create furrows in the underlying substrate that guide following cells towards the leading edges of the biofilm. They also found that extracellular DNA (eDNA) is crucial for this behaviour; in the presence of DNaseI, formation of the network was inhibited, a significant proportion of cells became stationary, and those that retained motility lacked coordinated movements. Thus, eDNA facilitates efficient trafficking of cells and is particularly important for coordinating assembly of the advancing cell aggregates at the leading edges, which forge into neighbouring trails and generate a highly interconnected network.