During spring 2010, four cases of bacteraemia caused by a tobramycin resistant Staphylococcus aureus (TRSA) of spa type t084 were identified in the neonatal intensive care unit at Ryhov County Hospital, Jönköping, Sweden. Retrospective investigation and screening revealed an outbreak of TRSA t084 that may have started in late 2009. This study describes the outbreak and the measures taken to eradicate it. Screening of infants, staff members, environmental samplings, antibiotic susceptibility testing and spa typing was done. Moreover, pasteurization and bacteriology screening of donated human milk, multimodal hygiene interventions, antibiotic substitution (tobramycin for amikacin) were done. TRSA septicaemia was statistically significantly more common before compared to after the antibiotic substitution in infants with TRSA in clinical samples. This may indicate an insufficient empirical antibiotic regimen. The S. aureus prevalence among the infants was reduced (p = 0.002) after the multimodal hygiene interventions, but had no effect on the proportion of TRSA. No TRSA was found after July 2019 to the end of follow-up in December 2021. Only S. aureus spa type t084 resulted in secondary spread, indicating that this strain may have virulence traits that improve colonization and spread. Hygiene interventions reduced the prevalence of S. aureus. After the antibiotic substitution, no more cases of TRSA septicaemia were observed. The outbreak strain was eliminated after five years. Antibiotic resistance surveillance systems may reveal the presence of resistant strains and facilitate early identification of an outbreak. Furthermore, it provides guidance for an effective empiric antibiotic regimen.