Introduction Vancomycin is the usual antibiotic treatment in coagulase-negative staphylococcus sepsis in premature infants but causes renal toxicity. As linezolid is effective in Gram-positive cocci infection, and devoid of renal side-effects, it has been used in Nantes neonatal intensive care units and linezolid plasma concentrations were monitored. Aim The aims of this study are to report data on linezolid concentrations in premature infants, describe clinical and bacteriological evolution during treatment, and determine potential side effects. Methods A retrospective observational study of premature infants treated with linezolid in Nantes Hospital from January 2008 through November 2011 was conducted. Linezolid plasma concentrations, possible side effects due to linezolid, and clinical response to linezolid treatment were collected from folder review. Results Twenty-four linezolid plasma concentrations were monitored in 16 premature patients, at steady state for continuous intravenous administration or 7 ± 1.5 h after last oral administration. Except for one case, linezolid plasma concentrations were ≥minimal inhibition concentration (MIC) for linezolid for both parenteral and oral administrations. We observed three cases of thrombocytopenia, two of leukopenia, three of neutropenia, and one of severe hyperlactacidemia, resolving after discontinuation of treatment. Clinical signs of infection resolved in 13/16 cases. Bacteria were coagulase-negative Staphylococci in 12/16 cases and were eradicated in 9/12 evaluable cases. Conclusions This study reports an adequate linezolid plasma concentration with regard to the linezolid MIC in extremely premature infants. However, considering adverse events reported, its use should be cautious and may concern only oral administration during the late phase of infection, to limit paradoxical catheter use to treat nosocomial infections. Moreover, safe and efficient anti-Staphylococcus therapies should be identified to treat this vulnerable population.