Microbial biofilms have been extensively studied for their roles in pathogenesis and are a source of panic in the medical sector. On the contrary, numerous non-pathogenic microorganisms form biofilms that can become a source for multitudinous bio-based chemicals that may have significant beneficial roles in therapeutics, industry farming and even livestock management. Microbial biofilms are a hub of biocatalyst-like compounds, and their property of adherence can help them sustain in continuous systems. However, detailed analysis of the compositions of microbial biofilms can be made possible by metabolomic studies that can determine precisely the metabolites present in the biofilm, the changes that occur due to micro-environmental alterations and the metabolites responsible for a specific sort of biocatalytic activity. Thus, in this chapter, the metabolome of different microbial biofilms and their metabolome study will be discussed with reference to their applications in medicine, industry and livestock management. This study can have humongous applications in the field of bio-based industries. The alterations in the dynamics of microbial biofilms due to changes in the microenvironment can be used to explore extremophilic biofilm formation. Stress application may trigger the production of a specific metabolite with a definite and interesting application in the aforementioned fields. A detailed study of the biofilm components can also have a fundamental role in designing new anti-biofilm agents, which can further help livestock disease and the medical sector in general. Therefore, this study aims to coalesce the beneficial aspects of microbial biofilms by delving deep into their metabolome.