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Simple Summary: This study aimed to investigate the impact of two different machine milking vacuum levels on the quantitative and qualitative characteristics of milk and the hygiene of mammary glands in ewes, when administered vitamin E and selenium (Se). The results indicated that the chemical composition of milk was not affected by the vacuum level, but the supplementation of vitamin E and Se had a positive influence on milk quality. Milk yield was notably increased with the 46 kPa vacuum level (high vacuum level). Furthermore, the supplementation of vitamin E and Se reduced the somatic cell count (SCC), which indicated better mammary gland health status at both vacuum levels. Significant differences in total bacterial count (TBC) between the vacuum levels were not observed, regardless of vitamin E and Se administration. In conclusion, the study suggests that increased vacuum level can improve milk yields and the supplementation of animal diets with vitamin E and Se has a positive impact on milk quality as well as on reducing SCC and improving the hygiene of machine milked ewes' mammary glands. The aim of this research was to study the effect of two machine milking vacuum levels on the quantitative and qualitative characteristics of milk and mammary gland hygiene of ewes, when vitamin E and Se were administrated supplementarily. The experiment was conducted at the Vlasti Research Station in the Greek province of West Macedonia. Ninety-six ewes of the Mountainous Greek sheep breed were used. Animals were separated in four equal groups of 24 ewes per group. A 2 × 2 factorial design was applied, with two milking vacuum levels (38 kPa and 46 kPa) and two rations, one supplemented with vitamin E (300 I.U.) and Se (3 mg/kg DM feed) and one without any vitamin E and Se supplementation. Six test days were assigned (evening and morning milkings) at 14-day intervals, from April to July. Following milk yield control, milk samples were collected for chemical composition and somatic cell count (SCC) determination. At the end of milking of each lot, the milk from the terminal receiver of the milking machine was received for the evaluation of total bacterial count (TBC). The results revealed that milk yield was improved considerably in the case of 46 kPa vacuum level. Moreover, the chemical composition of milk was not influenced by vacuum level; however, the administration of vitamin E and Se appeared to have a positive effect. Moreover, the addition of vitamin E and Se decreased somatic cell counts (number and log10) at the two assessed machine milking vacuum levels. In reference to TBC and their log10, significant differences were not observed at both milking vacuum levels, regardless of vitamin E and Se administration. Statistical analysis did not indicate any interactions between the factors that were studied. Therefore, it is concluded that the quantity of vitamin E and Se supplemented to the ration has a positive effect on decreasing SCC and consequent positive action in the hygiene of the mammary glands of machine milked ewes. [ABSTRACT FROM AUTHOR]