Queuosine (Q) is a modification of the wobble base in tRNAs that decode NAC codons. It is ubiquitous in bacteria, including many pathogens. Streptococcus mutans is an early colonizer of dental plaque biofilm and a key player in dental caries. Using a combination of genetic and physiological approaches, the predicted Q synthesis and salvage pathways were validated in this organism. These experiments confirmed that S. mutans can synthesize Q de novo through similar pathways found in Bacillus subtilis and Escherichia coli. However, S. mutans has a distinct salvage pathway compared to these model organisms, as it uses a transporter belonging to the Energy Coupling Factor (ECF) family controlled by a preQ ₁-dependent riboswitch. Furthermore, Q levels in this oral pathogen depended heavily on the media composition, suggesting that micronutrients can affect Q-mediated translation efficiency.