Picobirnaviruses are small double-stranded RNA viruses first discovered in 1988 in stool samples from patients with diarrhea. It has generally been assumed that picobirnaviruses infect animal hosts and that they are potential agents of diarrhea, but there is still no direct evidence demonstrating that picobirnaviruses infect animals. In the metagenomic era, virome studies have broadened our understanding of picobirnavirus genetic diversity and genome organization, expanded the types of animals in which they have been detected, and identified novel associations with human disease. Most importantly, from the wealth of new sequencing data and comparative genomic analyses, a provocative new hypothesis has emerged that picobirnaviruses may not infect animals, but rather that they may infect evolutionarily simpler denizens of the gastrointestinal tract: bacteria and/or fungi. Depending on whether the true hosts of picobirnaviruses are animals, fungi, or bacteria, the mechanisms by which they impact animal biology will vary dramatically.