The objective of this study was to develop and assess the efficacy of an aggressive deep cleaning sanitation standard operating procedure (DC-SSOP) in nine retail delicatessens to reduce persistent Listeria monocytogenes environmental contamination. The DC-SSOP was developed from combined daily SSOPs recommended by the Food Marketing Institute and input from experts in Listeria control from food manufacturing and sanitation. The DC-SSOP was executed by a trained professional cleaning service during a single 12-h shutdown period. A modified protocol from the U.S. Food and Drug Administration Bacteriological Analytical Manual was used to detect L. monocytogenes in samples from 28 food and nonfood contact surfaces that were collected immediately before and after each cleaning and in samples collected monthly for 3 months. The DC-SSOP significantly reduced L. monocytogenes prevalence overall during the 3-month follow-up period and produced variable results for persistent L. monocytogenes isolates. Six delis with historically low to moderate L. monocytogenes prevalence had no significant changes in the number of samples positive for L. monocytogenes after deep cleaning. Deep cleaning in very high prevalence delis (20 to 30% prevalence) reduced L. monocytogenes by 25.6% (Padj < 0.0001, n = 294) overall during the follow-up period. Among delis with extremely high prevalence (>30%), positive samples from nonfood contact surfaces were reduced by 19.6% (Padj = 0.0002, n = 294) during the follow-up period. The inability of deep cleaning to completely eliminate persistent L. monocytogenes was likely due to the diverse infrastructures in each deli, which may require more individualized intervention strategies.