ABSTRACT: Cistus ladanifer (rockrose) is a perennial shrub quite abundant in the Mediterranean region, and it is a rich source in secondary compounds such as condensed tannins (CTs). Condensed tannins from C. ladanifer were able to change the ruminal biohydrogenation (BH), increasing the t11-18:1 and c9,t11-18:2 production. However, the adequate conditions of the C. ladanifer CTs used to optimize the production of t11-18:1 and c9,t11-18:2 is not yet known. Thus, we tested the effect of increasing the doses of C. ladanifer CT extract (0, 25, 50, 75 and 100 g/kg dry matter (DM)) on in vitro rumen BH. Five in vitro batch incubations replicates were conducted using an oil supplemented high-concentrate substrate, incubated for 24 h with 6 mL of buffered ruminal fluid. Volatile fatty acids (VFAs) and long chain fatty acids (FA) were analyzed at 0 h and 24 h, and BH of c9-18:1, c9, c12-18:2 and c9, c12, c15-18:3, and BH products yield were computed. Increasing doses of C. ladanifer CTs led to a moderate linear decrease (p < 0.001) of the VFA production (a reduction of 27% with the highest dose compared to control). The disappearance of c9-18:1 and c9,c12-18:2 as well as the production of t11-18:1 and c9, t11:18:2 was not affected by increasing doses of C. ladanifer CTs, and only the disappearance of c9, c12, c15-18:3 suffered a mild linear decrease (a reduction of 24% with the highest dose compared to control). Nevertheless, increasing the C. ladanifer CT dose led to a strong depression of microbial odd and branched fatty acids and of dimethyl acetals production (less than 65% with the highest dose compared to control), which indicates that microbial growth was more inhibited than fermentative and biohydrogenation activities, in a possible adaptative response of microbial population to stress induced to CTs and polyunsaturated fatty acids. The ability of C. ladanifer to modulate the ruminal BH was not verified in the current in vitro experimental conditions, emphasizing the inconsistent BH response to CTs and highlighting the need to continue seeking the optimal conditions for using CTs to improve the fatty acid profile of ruminant fat.