A Multiplex PCR-based assay (m-PCR) with three sets of primers was developed for the detection of all serotypes of Salmonella enterica and the identification of Salmonella Enteritidis and Salmonella Typhimurium. This method was evaluated against a bacteriological method for the analysis of environmental swabs of poultry houses. Samples were preenriched in phosphate-buffered peptone water for 24 h and subjected to three different protocols prior to PCR: (i) an immunomagnetic separation using Dynabeads anti-Salmonella (Dynal); (ii) a DNA extraction procedure using the Instagene matrix; (iii) an additional step of culture on an MSRV medium. With protocols 1 and 2, eight positive results were found by PCR and 20 with the bacteriological method. Protocol 3 combining MSRV and PCR gave similar results to those obtained from bacteriological methods and allowed Salmonella detection within 2 days.