For babies at risk of early-onset sepsis (EOS), empiric ampicillin and gentamicin therapy was suggested. All EOS pathogens had little antimicrobial susceptibility data. A retrospective evaluation of antimicrobial susceptibility data from a prospective EOS surveillance study of children delivered between 22 weeks and cared for in Neonatal Research Network sites between April 2015 and March 2017. Based on the outcome, nonsusceptible was defined as intermediate or resistant. In 235 EOS instances among 217,480 live-born newborns, researchers found 239 pathogens (235 bacteria, 4 fungi). Antimicrobial susceptibility data was provided for 189/239 (79.1%) of the isolates. In vitro susceptibility testing revealed that all 81 Gram-positive isolates having ampicillin and gentamicin susceptibility data were susceptible to either ampicillin or gentamicin. 72/94 (76.6%) Gram-negative isolates having ampicillin and gentamicin susceptibility data were nonsusceptible to ampicillin, 8/94 (8.5%) isolates were nonsusceptible to gentamicin, and 7/96 (7.3%) isolates were nonsusceptible to both antibiotics. Only 5% of Gram-negative isolates examined were resistant to third and fourth generation cephalosporins, piperacillin-tazobactam, and carbapenems. Investigators estimated that isolates resistant to both ampicillin and gentamicin caused 8% of EOS cases; they were most likely to occur in preterm, very-low-birth-weight infants. The combination of ampicillin and gentamicin was effective against most modern EOS pathogens. Clinicians might consider adding broader-spectrum medication to neonates at high risk of EOS. Still, they advised that substituting or adding a single antimicrobial drug was unlikely to offer appropriate empiric therapy in all situations.