AbstractCRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because they are difficult to identify, the natural diversity and impact of Acrs on microbial ecosystems is underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection that isolatesacrgenes based on their ability to inhibit CRISPR-Cas function. Using this selection, we discovered ten DNA fragments from human oral and fecal metagenomes that antagonizeStreptococcus pyogenesCas9 (SpyCas9). The most potentacrdiscovered,acrIIA11, was recovered from aLachnospiraceaephage and is among the strongest known SpyCas9 inhibitors.AcrIIA11homologs are distributed across multiple bacterial phyla and many divergent homologs inhibit SpyCas9. We show that AcrIIA11 antagonizes SpyCas9 using a different mechanism than that of previously characterized inhibitors. Our study highlights the power of functional selections to uncover widespread Cas9 inhibitors within diverse microbiomes.