Biofilms, complex microbial communities embedded in extracellular polymeric substances (EPS), represent a significant challenge in both clinical and industrial settings due to their high resistance to antimicrobial agents. Understanding biofilm architecture is crucial for developing effective treatment strategies. Biomedical imaging techniques are essential tools for visualizing biofilms and investigating their structural and functional characteristics. Techniques such as light microscopy, Raman microscopy, electron microscopy, optical coherence tomography and magnetic resonance imaging have been extensively utilized in biofilm studies. CLSM allows detailed three-dimensional visualization and quantitative analysis, Raman microscopy provides molecular insights without invasive preparation, while electron microscopy offers high-resolution structural information. OCT enables real-time, non-invasive monitoring of biofilm growth, and MRI reveals metabolic and structural dynamics. This paper reviews these biomedical imaging technologies, highlighting their advantages, limitations, and their specific applicability in biofilm research, ultimately aiming to facilitate improved therapeutic interventions against biofilm-associated infections.