Comprehension of underlying mechanisms of probiotic action will support rationale selection of probiotic strains and targeted clinical study design with a higher likelihood of success. This will consequently contribute to better substantiation of health claims. Here, we aim to provide a perspective from a microbiology point of view that such comprehensive understanding is not straightforward. We show examples of well-documented probiotic effector molecules in Lactobacillus and Bifidobacterium strains, including surface-located molecules such as specific pili, S-layer proteins, exopolysaccharides, muropeptides, as well as more widely produced metabolites such as tryptophan-related and histamine-related metabolites, CpG-rich DNA, and various enzymes such as lactase and bile salt hydrolases. We also present recent advances in genetic tool development, microbiome analyses and model systems, as well as perspectives on how the field could further progress. This opinion is based on a discussion group organized at the annual meeting of the International Scientific Association on Probiotics and Prebiotics (ISAPP) in June 2017.