Antibiotic resistance is a major health challenge globally. Therefore, there is the need for new antibiotics. Here, we screen compounds with new scaffolds using nuclear magnetic resonance (NMR) spectroscopy and identified compounds that bind weakly to a 27-nucleotide (nt) construct of the bacterial 16S ribosomal RNA (rRNA). We show that, at high concentration, these compounds interfere with bacterial growth. We also observed that the use of efflux pump inhibitors in combination with the fragments enhanced their antibacterial properties and may help combat multidrug resistance. In addition, using a chemoenzymatic isotopic labeling strategy, we observed by NMR a slight modification of the motions within the bacterial rRNA in complex with one of the compounds. We further made use of various isotope labels for the partial resonance assignment of the human cytoplasmic 18S rRNA and probed its dynamics. Lastly, we demonstrated the use of 19F NMR for characterizing the structure of the trans-activation responsive RNA element of the HIV-2 promoter and human hepatitis B virus encapsidation signal epsilon element.