Tannins are phenolic compounds that interfere with biohydrogenation (BH) of polyunsaturated fatty acids (FAs). The aim of the present in vitro study was to investigate the effects of two different sources of tannins on FA profiles of rumen bacteria, with particular reference to rumenic and vaccenic acid. A control diet (C; composed of 300 g/kg of wheat straw, 132 g/kg of soyabean meal, 96 g/kg of barley meal, 152 g/kg of maize meal, 300 g/kg of maize gluten and 20 g/kg of mineral vitamin premix, all expressed on dry matter (DM)) and four diets, obtained by adding to C two different types of tannins from chestnut (TC) and from quebracho (TQ) at two concentration levels (49 and 82 g/kg DM), were compared. The content of the main unsaturated FAs (C18:1 cis9, C18:1 trans11, C18:2 cis9, cis12 and C18:3 cis9, cis12, cis15) from solid-associated bacteria (SAB) and liquid-associated bacteria (LAB) was affected by the presence of tannins in the diets. In particular, C18:1 trans11 content was significantly increased, especially with TC1, whereas the decreasing of C18:1 cis9 was unaffected, regardless of the presence or the kind of tannins added to feeds. SAB contained higher amounts of intermediates of polyunsaturated FA BH (as C18:1 trans11 and C18:2 cis9, trans11) than LAB that were characterized by a higher amount of C18:0. In the concentration range adopted in this study, the effect of TC and TQ on changes of bacterial FA profile was comparable. Tannins seem to be a good means to modulate the FA profile of rumen bacteria, favouring the accumulation of C18:1 trans11 during in vitro rumen fermentation.