The aim of this study was to determine the effects of a medicinal plant mixture (MP), sunflower oil (SO) and a combination of the medicinal plant mixture and sunflower oil (MPSO) on fermentation end products, the fatty acid composition of the rumen fluid, the ruminal microbial population as well as antioxidant status in the blood of sheep fed a high-concentrate diet. Four rumen-fistulated rams were randomly assigned according to a 4 x 4 Latin square design and fed a basal diet consisting of 720g DM/day meadow hay and 540 g DM/day barley grain. The basal diet was supplemented with no additive (control), a medicinal plant mixture (MP, 10% replacement of meadow hay; 72 g DM/day), sunflower oil (SO, 36 g/day) and the combination (MPSO). Before the in vivo experiment, 24h ruminal incubations in vitro were performed (meadow hay/barley grain; 400/600, w/w; MP; 10% replacement of meadow hay; SO; 3.5% DM; MPSO). The in vitro experiment with MP and MPSO increased the in vitro dry matter digestibility (P < 0.001) and decreased methane production (P = 0.021), while the MPSO treatment increased the concentration of polyunsaturated fatty acids (PUFA; P < 0.001). In sheep, the rumen characteristics were not affected by the treatments. No treatment effect on the rumen eubacterial population was detected by the 165-polymerase chain reaction-denaturizing gradient gel electrophoresis method. The beneficial effects of MP and MPSO on PUFA concentration observed in vitro were not fully confirmed in vivo. However, these results could point to the promising effects of using a medicinal plant mixture in high-concentrate diets of ruminants without adverse effects on the fermentation characteristics and microbial ecosystem in sheep rumen. (C) 2015 Elsevier B.V. All rights reserved.