Consumption of over-the-counter probiotics for promotion of health and well-being has increased worldwide in recent years. However, although probiotic use has been greatly popularized among the general public, there are conflicting clinical results for many probiotic strains and formulations. Emerging insights from microbiome research enable an assessment of gut colonization by probiotics, strain-level activity, interactions with the indigenous microbiome, safety and impacts on the host, and allow the association of probiotics with physiological effects and potentially useful medical indications. In this Perspective, we highlight key advances, challenges and limitations in striving toward an unbiased interpretation of the large amount of data regarding over-the-counter probiotics, and propose avenues to improve the quality of evidence, transparency, public awareness and regulation of their use.