The bacterial transcription terminator Rho is a hexameric ATP-dependent RNA helicase that dislodges elongating RNA polymerases. It has an N-terminal primary RNA binding site (PBS) on each subunit and a C-terminal secondary RNA binding site at the central channel. Here, we show that Rho also binds to linear longer double-stranded DNAs (dsDNAs) and the circular plasmids nonspecifically using its PBS. However, this interaction could be competed efficiently by single-stranded DNA (ssDNA), dC34. Long dsDNA (3.5 kb) at the PBS activates short oligo C RNA-mediated ATPase activity at the secondary binding site (SBS). The pre-bound Rho to this long DNA reduces the rate and efficiency of its transcription termination activities in vitro. Elevated concentrations of Rho reduced the in vitro transcription level suggesting that Rho might also function as a nonspecific repressor of gene expression under certain conditions. In the mid-log phase culture, Rho molecules were concentrated at the poles and along the membrane. In contrast, the Rho hexamers were observed to be distributed over the bacterial chromosome in the stationary phase likely in a hyper-oligomeric state composed of oligomers of hexamers. We propose that Rho molecules not engaged in the transcription termination process could use the bacterial chromosome as a "resting surface". This way the "idle" DNA-bound Rho molecules could be kept away from accidentally loading onto the nascent RNA and initiating unwanted transcription termination.