Confocal Raman spectroscopy is a non-destructive analytical method which is useful to obtain detailed information about the molecular composition of biological samples. Its high spatial resolution was used to collect spectra of single basidiospores of macrofungi of the genera Collybia, Gymnopus, Laccaria, Lactarius, Mycena and Russula. These spectra can be divided into three major taxon-related groups, with general compositional differences, such as the relative amount of lipids compared to proteins. In this study, collapsing of thin-walled spores during storage was often observed, a phenomenon which has been given little attention in the literature. The Raman spectra are treated with different chemometric preprocessing techniques, including Savitsky-Golay, standard normal variate (SNV) preprocessing and extended multiplicative scatter correction (EMSC). By using linear discriminant analysis, approximately 90% of the spectra can be assigned to the correct genus, but identification on the species level was not possible.