Notwithstanding the National Salmonella Control Programs, the latest data published by the European Food Safety Authority show an increase in Salmonella Enteritidis prevalence in laying hen flocks. For this reason, the implementation of innovative techniques such as phage therapy is needed to control Salmonella at farm level. Most common bacteriophage applications are a cocktail of 2 or more bacteriophages, as it has been described that cocktails could remove different Salmonella serotypes, thus providing cross efficacy. Nevertheless, resistance to the bacteriophage cocktail has been reported, resulting in a decrease in their effectiveness. Along these lines, some authors have reported the possibility of using autophage when commercial bacteriophage cocktails are not active against field strains. To our best knowledge, no autophage (bacteriophage isolated from the same environment where the pathogen is isolated) has been found to control Salmonella in laying hens. In this context, the aim of this study was to assess the application of autophage in reducing Salmonella Enteritidis in environmental and fecal samples in a layer farm. To this end, the bacteriophage was isolated from the same farm where the bacteria was present and was applied onto the facility installations and the animals, at 2 different times. After bacteriophage challenges, swab cloths from facility surfaces and feces samples were collected at 3 times according to the time spent after the bacteriophage challenge. The results obtained in our study showed that all the surface samples collected from the farm facilities after phage therapy were negative for Salmonella. Concerning faces samples, statistical differences were found in Salmonella counts, with the strongest decrease (1.78log10) occurring after the second challenge. Otherwise, depending of the moment of sampling, the results obtained were 2.34log10, 1.39log10, 0.56log10, and 0.97log10 cfu/g for T0, T1, T2, and T3 respectively. The study highlights the use of autophage therapy not only for Salmonella Enteritidis control in animals, but as a sanitizer in cleaning and disinfection.