A prospective study was performed in a paediatric hospital to evaluate the incidence of bacterial contamination in enteral nutrition bags and to determine the critical points of process. During two separate one-month periods, all children receiving pump-assisted enteral nutrition were enrolled in the study. Samples for microbiological analysis were collected from enteral nutrition bags after administration in the first and second study period (sample T₂). In the second study period, two additional samples were made at the end of the feed preparation process. One was refrigerated immediately (sample T₀) and the other was sealed in a tube that followed the enteral nutrition solution until the end of its administration (sample T₁). Bacterial contamination was detectable above 10²cfu/mL. Twenty-six out of 40 patients were included in the first study period and 14 out of 44 in the second study period. Contamination (>10²cfu/mL) occurred in nine of 26 samples (35%) and seven of 14 samples (50%) in the first and second study periods, respectively. Of these, five (20%) and three (21%) contained significant contamination (≥10⁴cfu/mL). Bacteria of low pathogenicity were found in T₀ samples. Bacteria present in T₂ samples were pathogenic and multiple in 50% of cases. These results suggest that manipulation of the enteral nutrition bags at the bedside is critical for bacterial safety.