Bacterial biofilms (BF) are surface-attached communities of cells embedded in an extracellular polymeric matrix. The cells are composed of polymers produced by the microorganisms themselves. Biofilm is a cause of multidrug resistance. A simple, reliable, quick biofilm analysis method facilitates the effective prevention and treatment of infectious diseases. In recent years, numerous methods for analyzing biofilm based on various principles have been developed. In this review, common microbiological, physical, and chemical methods of biofilm analysis are summarized, with a focus on the application of microscopy methods. New technologies such as Raman spectroscopy, imaging mass spectroscopy, and matrix-assisted laser desorption ionization-time of flight analysis are being applied in biofilm research. Accordingly, this review highlights the advantages and limitations of several methods with a view to assisting scientists in determining the most appropriate and up-to-date methods for their continued research.