The importance of extracellular DNA (eDNA) in biofilm formation has become increasingly clear from research on clinically relevant bacteria. This study aimed to determine whether the quantity of eDNA produced can be linked to the ability to form biofilm. We systematically quantified eDNA over time during planktonic growth and biofilm formation in Reinheimera sp. F8 and three other environmental isolates belonging to the genera Pseudomonas, Microbacterium and Serratia. eDNA in biofilms was visualised by fluorescence microscopy and quantified by PicoGreen® labelling without further sample preparation, whereas eDNA in planktonic cultures was precipitated before labelling and quantification. The effect of eDNA removal was investigated by DNase treatment. eDNA appeared in the early exponential growth phase of planktonic batch cultures and the concentration peaked in the stationary phase. The concentration in biofilms differed substantially between strains and over time during biofilm development. eDNA was important for the initial attachment in all strains, and DNase treatment reduced biofilm formation in three of four strains. The extent to which eDNA accumulated in planktonic cultures or biofilms did not reflect its significance to biofilm formation, and even very low concentrations of eDNA affected biofilm formation strongly. The significance of eDNA for biofilm formation in nature may thus be more widespread than previously anticipated.