Biofilms are complex microbial structures that are encapsulated by self-produced matrices and are considered as one of the most fascinating phenomena in the world of microbiology. The formation of biofilm primarily relies on the ability of adhesion of microorganisms on a substrate to form clusters and finally to develop an intricate structure. This structure plays a key role by providing media for communication, by absorbing nutrition and by protecting the microorganisms that reside inside. Thorough research has been dedicated to the elucidation of biochemical pathways that are involved in biofilm formation. These extensive investigations also established the understanding of attachment proteins, quorum sensing (QS) and extracellular polymeric substances (EPS). Research has also shed light on understanding the structural and functional aspects of microorganisms within biofilms which are versatile and dynamic. Microorganisms residing inside this complex architecture possess the ability for reciprocal coordination as well as competition for resources and niches which improve their organizational capacity to withstand adverse environmental conditions and antimicrobial agents. Besides, biofilm promotes microbial diversity and evolution by enhancing the flow of genes among microbes. Biofilm is an enigmatic architecture that provides a distinctive interface among biotechnology, microbial ecology and environment. Extensive research on their development, function, evolution and technological applications could clarify both the advantages and disadvantages of these microorganisms on human civilization and nature. This chapter highlights biofilm formation, its functional aspects, the relationship with the environment, its application in biotechnology, technical advancements in research and its negative influences.