This study reports the synthesis, antibacterial evaluation and nature of fluorine–rRNA contacts revealed by an X-ray co-crystal structure of a series of 4′-deoxy-4′-fluoro B-neomycin analogs. 4′-Deoxyfluorination improves the inhibition profile towards resistant enzymes and renders equally potent antibiotics compared to the parent neomycin B. The 4′-deoxy-4′-fluoro-4′-epi neomycin analogs showed a preferential inhibition over the 4′-deoxy-4′-fluoro neomycin counterpart against the strains of P. aeruginosa carrying a chromosomal APH(3′)-IIb enzyme, known to inactivate the parent aminoglycoside. To the best of our knowledge, this is the first example of a neighboring-group aminoglycoside-modifying enzyme evasion by fluorine substitution. A unique F-G1491 stacking was observed in a co-crystal structure of 4′-deoxy-4′-fluoro-4′-epi neomycin with a bacterial ribosomal RNA A-site.