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Although blood culture is considered the gold standard for detection of neonatal infections of bloodstream, its result can take from 48-72 h and has low sensitivity. In this study, we determined the usefulness of the panbacterial polymerase chain reaction (PCR) and the panviral RT-PCR in serum, as tests for the early diagnosis of neonatal sepsis. We studied 195 patients with the clinical diagnosis of neonatal sepsis, up to the age of 28 days, hospitalized in the Neonatal Intensive Care Unit (NICU) of the Hospital Infantil de México “Federico Gómez”, and 195 umbilical cord samples obtained from healthy newborns. Blood cultures, and DNA / RNA extraction were done with a system of primers of the rRNA 16s region, which is highly conserved in a large number of bacteria and a highly conserved non-coding enteroviral 5´ (5’-NCR) region, respectively. The results showed that among the 90 newborns with sepsis and negative PCR, 19 were randomly selected to perform RT-PCR; 26% of them had a positive result. Statistical analysis included the PCR test’s sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) using the clinical diagnosis as the gold standard. PCR sensitivity was 77%, specificity was 83%, PPV was 92% and the NPV was 59%. In summary, panbacterial PCR is useful, quick and effective in the diagnosis of sepsis and should be used as a complement to the microbiological and clinical diagnoses. Although the frequency of enteroviral sepsis was low, in newborns with a negative panbacterial PCR, a panviral RT-PCR is recommended. Our results indicate that molecular diagnosis provides the balance of cost-benefit.