The objective of our study was to assess factors associated with iatrogenic events in Neonatal Intensive Care Units (NICUs). This was a retrospective analysis based on a cohort of patients who participated in our previous prospective study (Pediatrics 122:550-555, 2008), conducted in four tertiary university-affiliated NICUs in Israel, that included all consecutive infants (n = 615) hospitalized during the study period. Ongoing monitoring of iatrogenic events was performed by designated "iatrogenesis advocates." The main outcome measures were the association of individual infant characteristics and NICUs' environmental characteristics with iatrogenic events assessed by univariate and multiple logistic regression analysis. We found that four infant characteristics were significantly (p < 0.001) associated with iatrogenic events in a univariate analysis: gestational age, birth weight, severity of initial illness as assessed by the Score for Neonatal Acute Physiology and Perinatal Extension (SNAPPE II), and length of stay (LOS). All four factors demonstrated a significant (p < 0.001) dose-response relationship with iatrogenic events. Univariate analysis for environmental characteristics showed that type of shift, but not nursing workload, was significantly associated with iatrogenic events (p < 0.001). In a multiple logistic regression analysis, only LOS (adjusted OR 1.02 [95 % CI, 1.01-1.03]) and type of shift, morning vs. evening (adjusted OR 3.44 [95 % CI, 2.33-5.08]) and morning vs. night (adjusted OR 6.07 [95 % CI, 3.86-9.56]), remained independently associated with iatrogenic events (p < 0.001). Prolonged LOS and morning shifts were found to be significantly associated with iatrogenic events. Further prospective research is warranted to identify the specific causes for iatrogenic events in order to target active interventions to prevent them.