Introduction: Insects have been described as mechanical vectors of nosocomial infections. Methods: Non-biting flying insects were collected inside a pediatric ward and neonatal-intensive care unit (ICU) of a Brazilian tertiary hospital. Results: Most (86.4%) of them were found to carry one or more species of bacteria on their external surfaces. The bacteria isolated were Gram-positive bacilli (68.2%) or cocci (40.9%), and Gram-negative bacilli (18.2%). Conclusions: Insects collected inside a hospital were carrying pathogenic bacteria; therefore, one must consider the possibility they may act as mechanical vectors of infections, in especially for debilitated or immune-compromised patients in the hospital environments where the insects were collected.Keywords: Microorganisms. Insecta. Public Health.Some non-biting flying insects, particularly the common housefly (Musca domestica), have sporadically been described as possible mechanical vectors of agents of nosocomial infections because they may carry human pathogens and also due to their ubiquity including in the hospital environments 1-4 .In Brazil, there are some reports showing that ants and cockroaches harbor pathogens in hospitals 5,6 , but there is no report on non-biting flying insects. Therefore, our aim was to investigate the presence of potentially pathogenic bacteria on the external surface of flying insects collected inside a Brazilian health care institution.Flying insects were randomly collected in eight rooms of a pediatric ward and a neonatal intensive care unit (neonatal-ICU) of a Brazilian tertiary-care teaching hospital that has 290 beds. The insects were caught during their flight during different nights. Each insect collected was placed alive on to a 5% sheep blood agar plate, allowing contact of its external surface with the culture medium. After removing the insect using sterile forceps, the plate was incubated at 37 o C under aerobic conditions and was examined daily for bacterial growth. The insect samples were preserved in alcohol and subjected to entomological examination.The morphology of isolated bacteria was observed using Gram staining. The Gram-positive cocci (GPC) or Grampositive bacilli (GPB) were separately cultured on sheep blood agar and the Gram-negative bacilli (GNB) on MacConkey agar for further identification in species by using standard bacteriological procedures.A total of 22 flying insects were collected: 5 (22.7%) were caught inside the neonatal-ICU and 17 (77.3%) in different rooms of the pediatric ward. They belonged to Diptera Linné,