Mare’s milk has always been appreciated due to its medicinal properties for which it has been more and more used in food, cosmetic and pharmaceutical industry and as a replacement for human milk in the diet of newborns. The aim of this study was to establish the chemical composition, physical properties and hygienic quality of mare’s milk of Croatian Coldblood horse breed. On the average, mare’s milk contained: 10.2 % total solids, 1.23 % milk fat, 1.76 % proteins, 0.71 % casein and 6.26 % lactose. The average pH value was 7.0, titratable acidity 2.51 °SH and freezing point -0.5318 °C. The total bacterial count was less than 58.000/mL and the somatic cell count 47x103/mL. The mare’s udder volume, environmental conditions and the presence of a foal (without a physical contact) showed a significant effect on the milk secretion. Based on the obtained results, criteria of physical-chemical and hygienic parameters of mare’s milk quality could be suggested. Possibilities of using the milk of Croatian coldblood horse breed are numerous, but until now it has almost not been used in Croatia due to the lack of information and knowledge of potential customers.