The aim of this study was to closely examine the Salmonella enterica serovar Enteritidis environmental contamination on persistently positive layer farms in Belgium during successive laying cycles. All of the farms were required to vaccinate their layers under the national control program for Salmonella. Seven farms with previous or current Salmonella Enteritidis contamination were monitored during different stages of the laying period and after cleaning and disinfection (CD). Environmental samples, including from the equipment and vermin, were taken in the henhouse and egg-collecting area. Dilutions were performed to define the degree of Salmonella Enteritidis contamination. Eggshells, egg contents, and ceca were also tested for Salmonella. At the end of the first sampled laying period, 41.6% of the environmental samples were contaminated with Salmonella Enteritidis. After CD, the prevalence dropped to 11.4%. On average, the prevalence in the second laying period increased again: 17.8, 18.4, and 22.3% at the onset, middle, and end of the lay period, respectively. After CD before the third laying period, the prevalence decreased to 6.6% and stabilized at the onset of lay (6.3%). During lay, as well as after CD, a wide variety of contaminated environmental samples were found; for example, in the henhouse, in the egg-collecting area, on mobile equipment and in or on vermin. In the henhouse during laying, the most recurrent and highly contaminated sites were the overshoes, floor, manure belt, and hen feces. The egg-collecting area had a significantly higher number of contaminated samples compared with that of the henhouse. For both sites, the floor appeared to be the most suitable sampling site to estimate the Salmonella Enteritidis status of the farms. Eggshell and egg content contamination varied between 0.18 and 1.8% and between 0.04 and 0.4%, respectively. In total, 2.2% of the analyzed ceca contained Salmonella Enteritidis. This study revealed that Salmonella Enteritidis is present in the environment of persistently Salmonella Enteritidis-contaminated layer farms, demonstrated that in many cases Salmonella Enteritidis contamination was not eliminated after CD, and identified the egg-collecting area as a critical point on most farms.