The remarkable ability of Enterococcus to produce microbial communities, specifically biofilms, is a topic of interest in scientific research. Biofilms formed by Enterococcus species, are known to contribute to their survival in extreme environments and their involvement in persistent bacterial infections. The aim of this chapter is to provide a comprehensive understanding of the mechanisms underlying biofilm formation in clinically important species such as E. faecalis and the increasingly drug-resistant but less well-studied E. faecium. Enterococcus forms biofilms through a complex interaction between genes and virulence factors such as DNA release, cytolysin, pili, secreted antigen A, and microbial surface components that recognize adhesive matrix molecules (MSCRAMMs). Quorum sensing mediated by peptide pheromones targets gene expression and regulation and is essential for the coordination of biofilm formation. Furthermore, control over extracellular DNA (eDNA) release has been shown to be crucial for biofilm formation. In E. faecalis, autolysin N-acetylglucosaminidase and proteases such as serine protease and gelatinase are important players in this process, influencing biofilm development and virulence. The study of biofilm formation in Enterococcus can provide insights into the pathogenesis of opportunistic infections and their prevention and provide directions for future anti-biofilm therapeutic research.