Abstract The aim of the present study was to assess performance of waste stabilization ponds (WSPs) on the removal of Listeria spp. in Isfahan, Iran. A total of 104 samples were taken from eight sampling locations from influent and effluent of a wastewater treatment plant (WWTP). Sewage samples were analyzed for the presence of Listeria spp. using selective enrichment protocol. Listeria isolates were also identified by biochemical and polymerase chain reaction (PCR) amplification. Listeria spp. was enumerated by a three tube most probable number (MPN) for total coliform counts (TC), fecal coliform counts (FC), total suspended solids (TSS), and total dissolved solids (TDS). In total, 54/104 (51.92%), 49/104 (47.11%), 36/104 (34.61%), and 27/104 (25.95%) samples were positive for Listeria spp., L. monocytogenes, L. innocua, and L. seeligeri, respectively. The mean MPN/100 mL enumeration of L. monocytogenes for influent, anaerobic, facultative ponds 1, 2, 3, 4 and maturation ponds 1 and 2 were 21.54, 10.61, 8, 5.77, 4, 2.54, 1.38, and 0.46, respectively. The removal percentage of Listeria spp. in the anaerobic, facultative, and maturation ponds were 44.71, 76.5, and 81.4%, respectively. Results showed that the WSPs were able to decrease the Listeria spp. levels significantly, although unable to remove them completely.