Cyclic di-GMP is a second messenger found in almost all eubacteria that acts to regulate a wide range of functions including developmental transitions, adhesion and biofilm formation. Cyclic di-GMP is synthesised from two GTP molecules by diguanylate cyclases that have a GGDEF domain and is degraded by phosphodiesterases with either an EAL or an HD-GYP domain. Proteins with these domains often contain additional signal input domains, suggesting that their enzymatic activity may be modulated as a response to different environmental or cellular cues. Cyclic di-GMP exerts a regulatory action through binding to diverse receptors that include a small protein domain called PilZ, enzymatically inactive GGDEF, EAL or HD-GYP domains, transcription factors and riboswitches. In many bacteria, high cellular levels of cyclic di-GMP are associated with a sessile, biofilm lifestyle, whereas low levels of the nucleotide promote motility and virulence factor synthesis in pathogens. Elucidation of the roles of cyclic di-GMP signalling in biofilm formation has suggested strategies whereby modulation of the levels of the nucleotide or interference with signalling pathways may lead to inhibition of biofilm formation or promotion of biofilm dispersal. In this review we consider these approaches for the control of biofilm formation, beginning with an overview of cyclic di-GMP signalling and the different ways that it can act in regulation of biofilm dynamics.