Foodborne listeriosis is one of the most serious and severe foodborne diseases, with a high mortality rate worldwide. Listeria monocytogenes’ (Lm) ability to survive under a diversity of conditions makes it a threat for food safety. Soft and semisoft cheeses are common RTE foods that support survival and growth of Lm due to their high moisture content and favorable pH. The aim of the present study was to assess, after artificial inoculation, the growth potential (Δ) of Lm in vacuum packed RTE soft Greek Anthotyros cheese. Growth potential of Lm was determined according to the new version of “EURL Lm Technical Guidance Document on challenge tests and durability studies for assessing shelf life of ready-to-eat foods related to Lm”. The results of our study revealed that the growth potential of Lm was (Δ) = 4.93 log10 CFU/g, indicating that the specific soft cheese “anthotyros” is a “Ready to eat food able to support growth of Lm and classified in food safety category 1.2 in the (EC) Regulation 2073/2005”. That means that the food business operator (FBO) must comply with the criteria that define that the bacterium must not be detected in 25 g (n = 5, c = 0) at the time of leaving the production plant. Through this study, we try to highlight the need for FBO to conduct relevant research to ensure that the ready-to-eat products which they produce, during their shelf life, do not support the growth of Lm.