In this study, the basic chemical and fatty acid compositions of autochthonous Croatian cow and sheep cheeses in a lamb skin sack (local name: “sir iz mišine”) were determined. Also, the influences of ripening period and different starter cultures on chemical and fatty acid composition of these cheeses were investigated. Samples of cow (n=20) and sheep (n=20) milk cheeses were produced in three different ways: from raw milk without the addition of a starter culture, from pasteurized milk with commercial starter cultures and with previously isolated autochthonous starter cultures (Lactococcus lactis S1 or Lactobacillus plantarum B or a mixture of both). Samples were taken during a 45-day ripening period (on days 0, 15, 30 and 45). The ripening time significantly affected all basic chemical parameters, while different starter cultures significantly (p<0.05) influenced protein, fat and ash content. Ripening time had no significant effect on the representation of the investigated fatty acid groups (p>0.05), but a significant difference was found depending on the starter cultures used and the type of cheese analysed, in terms of statistically higher proportion (p<0.05) of polyunsaturated fatty acids in the finished sheep's milk cheese (2.58–2.97%) in comparison to the cow's milk cheese (1.93–2.14%). Fatty acids most represented in the analysed cheeses were palmitic, oleic and stearic acid.