Food safety, within the context of the hospital's Food and Nutrition Units (FNU), is one of the key factors in ensuring the quality of the service. Thus, the aim of this study was to analyze the microbiological contamination of sponges and multipurpose cloths used in a hospital's FNU, as well as the effectiveness of different disinfection methods. The materials were submitted to viable cell count for total and thermotolerant coliforms, coagulase-positive Staphylococcus, and mesophiles; in addition, efficiency tests of the disinfection methods carried out by the FNU were done by scanning electron microscopy, antimicrobial susceptibility testing, biofilm formation capacity, and virulence factors. The analysis results confirmed the contamination of these utensils, and the disinfection methods used were efficient. Electromicroscopic analysis identified high bacterial adhesion after use, and the isolates were resistant to the antimicrobial penicillin and classified as poor biofilm producers. Sponge isolates showed greater virulence than other materials. Therefore, good practices in food services must follow strict hygiene and disinfection protocols to ensure the safety of the food produced.