Optical cathodoluminescence (CL) is commonly used to identify diagenetically altered carbonate fossils, yet such an interpretation is problematic as present-day carbonate shells may also luminesce. Hyperspectral CL imaging combines CL microscopy and CL spectroscopy to quantitatively analyze luminescence emission. Cold optical CL and hyperspectral CL imaging were carried out on four modern biominerals, a Rhynchonelliform brachiopod, a Craniid brachiopod, a bivalve, and the eggshell of the domestic fowl. A fossil Craniid brachiopod was analyzed to compare luminescence emission with that from the modern Craniid brachiopod. The beam conditions used for optical CL vary between studies, which hinders the direct comparison of CL analyses. This study assesses the effect of beam current and beam diameter on the intensity of luminescence emission. By characterizing the effect of beam conditions on different CaCO3 biominerals, comparisons can be made between CL studies. Hyperspectral CL imaging can be carried out in combination with WDS element analysis. By comparing hyperspectral CL images with element maps the causes of luminescence can to some extent be determined. The intensity of luminescence emitted from the modern biominerals differs under the same beam conditions. All four modern shells emit blue luminescence. In N. anomala, there is a correlation between Mn2+ concentration and luminescence intensity in the 620- to 630-nm wavelength band, which is apparent in the inner region of the shell. The fossil Craniid also emits blue luminescence, and texture within the shell wall is apparent; however, the luminescence emission between 620 and 630 nm that is evident in N. anomala is absent.