Objectives: The discovery of new antibiotic targets is important to stem the increase in antibiotic resistance to most currently used antimicrobials. The bacterial ribosome is a major target for a large number of antibiotics that inhibit different aspects of translation. Most of these antimicrobial agents also inhibit ribosomal subunit formation as a second cellular target. Precise subunit assembly requires the activity of several distinct RNases for proper rRNA processing. The present work shows that the vanadyl ribonucleoside complex (VRC) inhibited RNases in Staphylococcus aureus involved in ribosomal subunit formation without an effect on translation. Methods: Methicillin-susceptible and -resistant strains of S. aureus were examined for the inhibitory effects of VRC on cell viability by colony counting. Protein synthesis rates were measured by isotopic methionine incorporation. Ribosome synthesis was measured by radiolabelled uridine incorporation into ribosomal subunits as displayed on sucrose gradients. Pulse and chase radiolabelling was used to measure subunit synthesis rates. RNA turnover was determined by a gel on a chip assay. Results: The rates of subunit synthesis and the amounts of both subunits were significantly reduced in the presence of the compound. Ribosomal RNA was degraded and cell viability was reduced as a consequence. VRC also stimulated the inhibitory effects of a macrolide and an aminoglycoside antibiotic on ribosome formation. Conclusions: Bacterial ribosomal subunit synthesis was specifically impaired in VRC-treated cells, with the rates and amounts of both subunits reduced. Cell viability was significantly reduced and rRNA turnover was stimulated.