16S rRNA methyltransferases have emerged as critical elements of high-level aminoglycoside resistance in clinical pathogens. We investigated the fitness costs associated with the expression of six methyltransferases isolated from clinical strains (ArmA, RmtA, RmtB, RmtC, RmtD, and NpmA), and two methyltransferases from natural antibiotic producers (Sgm and KamB) in . Growth competition assays revealed that methyltransferases found in natural producers imposed significantly lower fitness costs than those isolated from clinical strains, allowing resistant populations to persist at stable levels. Translational fidelity assays demonstrated that most methyltransferases induce error-prone phenotypes by allowing increased readthrough of nonsense codons and frameshift mutations, while KamB uniquely increased translational accuracy. Deletion of the housekeeping methyltransferase RsmF further altered these effects, highlighting the complex interplay between endogenous and exogenous methylation processes. Stress response experiments showed varying results: most methyltransferases increased susceptibility to hyperosmotic stress, while several (RmtB, RmtA, ArmA, and KamB) increased tolerance to acidic stress. These findings reveal that 16S rRNA methyltransferases play complex roles in bacterial physiology beyond antibiotic resistance, with important implications for the persistence of resistance and potential therapeutic strategies targeting specific vulnerabilities in resistant bacteria.