Microbial cells favor colonial behavior to survive and multiply under harsh environmental conditions. It constructs a well-defined biofilm structure accommodating multiple microbial cells aggregated on biotic and abiotic surfaces. The microbial cells within the matrix are resistant and adaptive to antibiotics compared to planktonic cells. Microbial cells are adhered to and embedded in a microbial matrix of microbial secretions, such as exopolysaccharides, lipids, and nucleic acids. Microbial biofilm creates a physical barrier to prevent antimicrobial penetration, increase resistance, and create resilient dormant cells. It helps evade local immune responses that can lead to a chronic infection. Gram-negative bacteria display more substantial mass spectra peaks than Gram-positive bacteria, as a multilayered peptidoglycan layer blocks lipid ionization. Lipidomics can be used as an effective tool to explore the lipid interactions within the communities, further attributing to their functional role, cellular architecture, functional membrane microdomains (FMMs), and regulating membrane protein-lipid interactions. This chapter provides detailed information regarding microbial lipid profiles, methodologies used, and their contribution to environmental stress and antibiotic resistance via lipidomic analysis. However, a limited amount of literature is available regarding microbial lipidomic analysis, which would be a great approach to analyze microbial lipid profiles with specific biomarkers to address the issues associated with infection biology using MALDI-TOF, ESI-MS, GC-MS, LC-MS, and NMR.