Streptomycetes are multicellular filamentous microorganisms, which are major producers of antibiotics, anticancer drugs and industrial enzymes. When grown in submerged cultures, the preferred enzyme producer, Streptomyces lividans, forms dense mycelial aggregates or pellets, which requires the activity of the proteins encoded by the matAB and cslA-glxA. Here we show that matAB encodes the biosynthetic genes for the extracellular polymeric substance (EPS) poly-β-1,6-N-acetylglucosamine or PNAG. Heterologous expression of matAB in actinomycetes that naturally lack these genes was sufficient for PNAG production and induction of mycelial aggregation. Also, overexpression of matAB in a non-pelleting cslA mutant restored pellet formation, which could effectively be antagonized by the PNAG-specific hydrolase, dispersin B. Extracellular accumulation of PNAG allowed Streptomyces to attach to hydrophilic surfaces, unlike attachment to hydrophobic surfaces, which involves a cellulase-degradable EPS produced by CslA. Altogether, our data support a model in which pellet formation depends on hydrophilic interactions mediated by PNAG and hydrophobic interactions involving the EPS produced by CslA. These new insights may be harnessed to improve growth and industrial exploitation of these highly versatile natural product and enzyme producers.