The objective of this experiment was to evaluate the effects of different feed additives (cinnamaldehyde, monensin, and quebracho condensed tannin extract) on fermentation, trans fatty acids (FA) formation and selected strains of rumen bacteria. Four continuous culture systems were used in 4 x 4 Latin square designs with 4 periods of 10 days each. Treatment diets were: control diet (44:56 forage to concentrate; CON), control plus cinnamaldehyde (CIN) at 400 mg/L, control plus monensin (MON) at 12 mg/L, and control with quebracho condensed tannin extract (QTAN) at 100 g/kg of diet (DM basis). Fermenters were fed treatment diets three times daily at 120 g/day and overflow (effluent) samples were collected from each fermenter on days 8, 9 and 10 of each period to estimate nutrients digestibility and FA composition. On day 10 of each period, three samples were collected from each fermenter at 3 and 6 h post morning feeding for volatile fatty acids (VFA), ammonia-N and bacterial analyses. Compared with the CON diet, feed additives had no effects (P > 0.05) on apparent dry matter (DM), neutral detergent fiber (NDF) and organic matter (OM) digestibility but apparent protein digestibility decreased (P < 0.01) with the QTAN and CIN diets. Compared with the CON diet, the concentration of acetate decreased (P < 0.05) with the MON and CIN diets. The concentration of propionate increased (P < 0.05) with the MON and QTAN diets and was greatest with the MON diet. Ammonia-N concentration decreased (P < 0.01) with all feed additives and was least with the QTAN diet. The concentration of C18:0 decreased (P < 0.01) with the three feed additives and was least with the MON diet. Concentration of trans C18:1 and vaccenic acid (VA) increased (P < 0.05) with the MON and CIN diets and was greatest with the MON diet. Compared with the CON diet, the concentration of c9t11CLA increased (P < 0.05) only with the QTAN diet. The DNA abundance of Butyrivibrio proteoclasticum decreased (P < 0.05) with the MON and CIN diets while the DNA abundance for Butyrivibrio VA increased (P < 0.05) with all feed additives compared with the CON diet. Feed additives had no effects (P > 0.05) on the DNA abundance of Anaerovibrio lipolytica and Butyrivibrio SA. In conclusion, results demonstrate that the feed additives used in this study affected the fermentation and biohydrogenation process. Addition of feed additives reduced the formation of C18:0 but only MON and CIN increased VA formation. MON and CIN effects on VA formation may in part be explained by their effects on B. proteoclasticum. (C) 2015 Elsevier B.V. All rights reserved.