Biofilms are common in nature and are an essential way for bacteria to survive in hostile environments. They can be advantageous or harmful, especially if they are created on medical equipment or in industrial environments. As a result, research on Biofilm production and removal is highly relevant to a variety of fields, including the food, medical, dentistry, and water quality sectors. Research on Biofilm development is moving more slowly than Biofilm detection techniques, which are still being improved. However, over the last 20 years, advancements have been achieved in several methods, including piezoelectric sensors, bioluminescent assay, microplate culture, and different microscopic analyses. Our knowledge of the physiology, composition, and structure of Biofilms has improved as a result of these approaches. Efficacy testing procedures for those creating anti-Biofilm surfaces in constructed settings need to be pertinent, repeatable, and consistent. This guarantees that data outputs are similar throughout the literature and suitable for end use. The most often utilized approaches involve the growth of Biofilm in both static and submerged systems, and the quantification methods include fluorescence, spectroscopy, microscopy, direct agar contact, sequencing, ELISA (Enzyme-linked Immunosorbent Assay), and colony-forming unit counts. The study of Biofilm biomass, viability, structure, composition, and physiology is covered in this chapter using both conventional and innovative approaches. It draws attention to the lack of agreement among the many methods for cultivating and researching Biofilms and offers a critical viewpoint on their benefits and drawbacks. Lastly, the possibility of using sophisticated microscopy techniques in microbial systems in the future is investigated. This is especially true when paired with other characterization techniques like Raman spectroscopy and microscopy to clarify the dynamic cellular processes. The goal of this thorough review chapter is to help researchers further their Biofilm study and create efficient anti-Biofilm tactics.