OBJECTIVE To evaluate the marginal cost of therapeutic failure with Ampicillin/amikacin as the first-line antibiotic treatment of early-onset neonatal sepsis (ENS). METHODS Out of a total of 121 newborns, 86 failed to respond to Ampicillin/amikacin when it was used as first-line treatment within their first 72h of life. All of them were admitted to the NICU between 2001 and 2005 with suspicion of sepsis. After this failure to respond, vancomycin and/or cefotaxime were used as the second treatment option for these newborns. Using a full cost method we performed a cost analysis with an activity-based-costing (ABC) perspective, identifying the costs generated by these 86 patients. RESULTS The costs comprising medication, hospitalization, supplies and clinical staff salaries were higher when vancomycin and/or cefotaxime were used (350,924 dollars or 275,116 euros) compared to those generated with an Ampicillin/amikacin regimen (159,251 dollars or 124,878 euros). When compared as protective factors, the relative risk was 1.09 (95% CI; 0.39-2.1 P=0.8) for Ampicillin/amikacin vs. 0.02 (95% CI; 0.04-0.32 P<0.05) for vancomycin and/or cefotaxime. CONCLUSIONS Even though vancomycin and/or cefotaxime are initially more costly, there is a cost saving derived from the use of this antibiotic treatment as the first-line therapeutic option instead of as a rescue therapy when a lower-efficacy regimen (Ampicillin/amikacin) has failed.