Tsalafouti cheese, a traditional Greek spreadable cheese, has experienced remarkable popularity over the last decade due to the strong dairy production heritage links that it presents, alongside the special nutritional and sensory attributes that it holds. The aim of this study was to chemically characterize the Greek cheese Tsalafouti, with a special focus on its fatty acid profile and the potential nutritional effect it may have. Standard methods were used to assess the physicochemical profile of Tsalafouti cheese, while a fatty acid profile was determined using gas chromatography. The analysis revealed average values, including a pH of 3.94, moisture content of 75.05%, protein content of 8.86%, and fat content of 11.3%. The fatty acid profile highlighted the predominance of health-important fatty acids, such as oleic acid, known for its well-established health benefits. Additionally, the health lipid indices of Tsalafouti were assessed by calculating the atherogenicity index (IA), the thrombogenicity index (IT), the ratio of hypocholesterolemic to hypercholesterolemic fatty acids (HH), the health-promoting index (HPI), and the PUFA-n-6/PUFA-n-3 ratio. The values of these indices indicate the quality concerning proatherogenicity, prothrombogenicity, and the risk of cardiovascular issues. Notably, all the above health indicators are consistent with known health effects. Importantly, the ratio of ω6/ω3 fatty acids aligns with the nutritional recommendations of the World Health Organization. Based on these results, Tsalafouti cheese emerges as a low-fat product and source of protein with potential functional properties, distinguishing itself from other cheeses in the market.