Like other aminoglycosides, netilmicin binds to bacterial ribosomes and blocks the protein synthesis, which has bactericidal consequences. It is an antibiotic with a broad spectrum with respect to Gram-negative bacili. All enterobacteriaceae (E. coli, Klebsiella, Enterobacter, Serratia, Proteus, etc.) are sensitive. Its activity against Pseudomonas aeruginosa is particularly useful. Netilmicin is also active against Staphylococcus aureus; however, methicillin-resistant staphylococci are often also resistant against aminoglycosides. Anaerobic germs are also resistant.

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