A multivariate image is an image stack in which each pixel contains several variables. Such images are common in many fields (medicine, imaging microscopy, satellite imaging...) and their analysis requires adapted multivariate statistical methods. In fluorescence imaging microscopy, different probes or different measurements such as intensity, fluorescence lifetime or spectral information can be observed from one view. However, this is not yet analysed as multivariate images. Here, we are presenting a full approach of multivariate analysis of fluorescence microscopy images and we are proposing a free R package (multifluo) to conduct it.