Newborn babies admitted into the neonatal intensive care unit (NICU) often require many supportive invasive devices and frequently receive antimicrobial therapy. We investigated the microbial flora in NICU patients reporting the distribution of infections in different catheter sites. Results showed that 97% of samples were positive; in particular 11% were positive for two or more microbial agents. Coagulase negative Staphylococci were the most commonly isolated. The detection of Gram-negative bacteria and yeasts suggested that these microorganisms are also involved in infections of hospitalized infants. Finally, no correlation between a specific microbial agent and a particular catheter type was found.