Bacterial RNA helicase rho is a genome sentinel that terminates the synthesis of damaged and junk RNAs that are not translated by the ribosome. It is unclear how rho is regulated during dormancy or stress, when translation is inefficient and RNAs are vulnerable to rho-mediated release. We use cryogenic electron microscopy, biochemical, and genetic approaches to show that substitutions of residues in the connector between two rho domains or ADP promote the formation of extended Escherichia coli rho filaments. By contrast, (p)ppGpp induces the formation of transient rho dodecamers. Our results demonstrate that ADP and (p)ppGpp nucleotides bound at subunit interfaces inhibit rho ring closure that underpins the hexamer activation, thus favoring the assembly of inactive higher-order oligomers. Connector substitutions and antibiotics that inhibit RNA and protein syntheses trigger rho aggregation in the cell. These and other recent data implicate aggregation as a widespread strategy to tune rho activity.