Confocal microscopy has emerged as a powerful imaging technique for studying cell mechanics and disease pathology, offering high-resolution, three-dimensional visualization of cellular structures and interactions. This chapter explores the diverse applications of confocal microscopy in biomedical research, with a particular focus on its role in investigating cellular mechanics and tissue engineering. By enabling detailed images of cytoskeletal organization, focal adhesions, and nuclear architecture, confocal microscopy provides critical insights into how mechanical forces influence cell behavior in both normal and engineered environments. Furthermore, the integration of confocal microscopy with advanced techniques, such as atomic force microscopy and particle tracking microrheology, has expanded its diagnostic and therapeutic potential. From analyzing scaffold architecture and extracellular matrix deposition to evaluating cell–scaffold interactions and tissue regeneration, confocal microscopy continues to enhance our understanding of tissue formation and mechanobiology. As technological advancements drive further improvements in imaging speed and resolution, confocal microscopy is expected to play an even greater role in tissue engineering and biomedical research.