Polyphosphates (polyP) are polymers of inorganic phosphates joined by high-energy bonds to form long chains. These chains are present in all forms of life but were once disregarded as ‘molecular fossils’. PolyP has gained attention in recent years following new links to diverse biological roles ranging from energy storage to cell signaling. PolyP research in humans and other higher eukaryotes is limited by a lack of suitable tools and awaits the identification of enzymatic players that would enable more comprehensive studies. Therefore, many of the most important insights have come from single-cell model systems. Here, we review determinants of polyP metabolism, regulation, and function in major microbial systems, including bacteria, fungi, protozoa, and algae. We highlight key similarities and differences that may aid in our understanding of how polyP impacts cell physiology at a molecular level.