The aim of the study was to compare two sources of tannins on fatty acids (FA) composition in rumen. Treatments were (g tannins/kg diet as-feed-basis) as follows: (1) no supplemental tannin addition (CON), (2) addition of 13 g of oak bark extract (OAK), and (3) 4 g of tannic acid (TAN). The basal diet contained 55:45 forage to concentrate ratio. Net consumption of tannins (g/d) was 4 g for both tannins sources. The study was performed on three Polish Mountain ewes fitted with rumen cannulas, and was divided into three experimental periods (I, II, and III). Both sampling time and animal diet had a significant effect on FA profile in the rumen fluid. In general, FA concentrations were higher before feeding in comparison to samples collected 2 and 4 h after feeding. In terms of dietary effect, it was shown that TAN addition had a greater influence on FA profile in the ruminal fluid than the OAK diet. Briefly, in the TAN group significantly increased concentrations of C18:2 (linoleic acid, LA) 8 h after feeding (vs. control, CON and OAK), C18:3 (α-linolenic acid, LNA) 4 h after feeding (vs. OAK), C20:3 n-6 before feeding (vs. CON), C20:4 before feeding (vs. CON and OAK) and 8 h after feeding (vs. OAK) were recorded. In contrast, OAK addition significantly reduced C20:3 n-6 concentration 2 h after feeding (vs. CON). In conclusion, increased concentrations of both LA and LNA in the rumen indicated that supplemental tannic acid may inhibit the initial stage of FA biohydrogenation in the rumen.