Microbial biofilms are complex bacterial communities, adhered to the surfaces and enclosed in an extracellular matrix. Biofilms are significant in different sectors, such as clinical, environmental, and industrial as they exhibit resistance to external stressors, such as immunological reactions and antimicrobial drugs. This chapter deals with the formation, physiology, and ecological significance of biofilms and focuses on their effect on industry and human health. Metagenomics is a revolutionary method to understand the genetic diversity and functional profiling of biofilms. Metagenomics offers extensive analysis of microbial communities, circumventing the drawbacks of conventional culturing techniques and revealing important information about metabolic pathways, resistance mechanisms linked to biofilms, and interspecies interactions. This chapter covers several metagenomic methods and their applications in biofilm studies including shotgun, whole genome, and 16S rRNA sequencing. Additionally, metagenomics has increased our understanding of biofilms in a range of environments, such as hospitals and archaeological sites. Metagenomics plays a pivotal role in the management of biofilm-related issues by providing cues about novel targets and developing effective anti-biofilm tactics.