Biogenic polyamines are natural aliphatic polycations formed from amino acids by biochemical pathways that are highly conserved from bacteria to humans. Their cellular concentrations are carefully regulated and dysregulation causes severe cell growth defects. Polyamines have high affinity for nucleic acids and are known to interact with mRNA, tRNA and rRNA to stimulate the translational machinery, but the exact molecular mechanism(s) for this stimulus is still unknown. Here we exploit that Escherichia coli is viable in the absence of polyamines, including the universally conserved putrescine and spermidine. Using global macromolecule labelling approaches we find that ribosome efficiency is reduced by 50–70% in the absence of polyamines and this reduction is caused by slow translation elongation speed. The low efficiency causes rRNA and multiple tRNA species to be overproduced in the absence of polyamines, suggesting an impact on the feedback regulation of stable RNA transcription. Importantly, we find that polyamine deficiency affects both tRNA levels and tRNA modification patterns. Specifically, a large fraction of tRNA^his, tRNA^tyr and tRNA^asn lack the queuosine modification in the anticodon “wobble” base, which can be reversed by addition of polyamines to the growth medium. In conclusion, we demonstrate that polyamines are needed for modification of specific tRNA, possibly by facilitating the interaction with modification enzymes.