Home-prepared enteral formulations are supplied to patients through enteral nutrition bottles, via a gravity bag or other container, which may be inadequately sanitized and reused more times than recommended by the manufacturer. Such procedures increase the risk of contamination and can compromise the patient's clinical outcome. In light of this, the present study aimed to assess the risk of contamination of enteral nutrition bottles by simulating home use conditions and hygiene procedures. A simulation of bottle usage was conducted across the three categories of enteral nutrition (homemade enteral preparations, blended enteral preparations, and commercial enteral formulas) for 3 days, using three hygiene procedures reported by caregivers: use of detergent (DET); use of detergent and boiling water (DET+BW); and use of detergent and bleach (DET+BL). The microbiological contamination was determined by the analysis of aerobic mesophilic microorganisms. The bottles that were used for 3 days, regardless of the enteral nutrition category, were within the acceptable limit for aerobic mesophilic microorganisms (between <4 and 8.0 colony-forming units [CFU]/cm2) when sanitized using the DET+BW and DET+BL procedures. The enteral nutrition bottles, when cleaned using the DET procedure during the 3 days of usage, showed low microbial contamination (between <4 and 3.0 CFU/cm2) in blended preparation and commercial formula only. Thus, regardless of the enteral nutrition category, we found that the bottles can be used for 3 days, as long as the DET+BW or DET+BL hygiene procedure is applied and safe food handling measures are adopted.