Confocal microscopy is nowadays one of the most extensively used tools for digital imaging. The advances in instrumental setup and fluorescence labelling strategies improve and broaden the possible experimental applications. Moreover, the implementation of solutions able to break the diffraction barrier coupled to confocal instruments brings up the study of the samples at super‐resolution level. This continuous evolution ensures confocal microscopy as a key platform in life science research. Key Concepts Components of confocal laser scanning and spinning disk microscopes. Fluorescence and fluorescent labelling tools. Confocal microscopy setup and main applications review. Confocal microscopy applications: reflection, subcellular localisation, dynamic studies and molecular interactions. Super‐resolution approaches in confocal microscopy.