To determine the effects of incremental quebracho tannin extract (QTE) infusions on rumen fermentation and yield and composition of microbial mass, a study was conducted that consisted of 1 control period (Control I) without QTE dosing; 4 periods when all animals received 1, 2, 4, or 6% QTE of the diet (DM basis); and a second control period (Control II). Each period consisted of 9 d of adaptation, 6 d of urine collection, and 1 d of rumen sampling. Three rumen-fistulated heifers (495 kg BW [SE 14]) were offered 2.6 kg/d grass hay, 2.6 kg/d concentrates, and 60 g/d mineral-vitamin premix (as fed) in 2 equal meals. Half the daily QTE dosage was intraruminally administered at every meal. Urine and rumen fluids were analyzed for purine derivatives and short-chain fatty acids, respectively. Nitrogen and purine base (PB) concentrations were determined in liquid-associated microbes in rumen fluid (LAMF), liquid-associated microbes in the solid phase (LAMS), and particle-associated microbes (PAM). Increasing QTE dosages linearly increased propionate and butyrate proportions in rumen fluid ( = 0.37, = 0.004 and = 0.51, < 0.001, respectively). Instead, proportions of acetate ( = 0.75, < 0.001), isobutyrate ( = 0.66, < 0.001), and isovalerate ( = 0.49, < 0.001) and urinary purine derivatives excretions ( = 0.66, < 0.001) linearly decreased with increasing QTE infusions. Inconsistent differences were observed for LAMF, but concentrations of PB in LAMS ( ≤ 0.018) and of N in PAM ( < 0.001) were greater at 6% QTE than at Control I, II, and 1% QTE. Hence, low to moderate QTE dosages hamper rumen fermentation and microbial biomass yields. Alongside the nutritional consequences for the host, the methodological implications of these effects should be considered in studies evaluating the targeted use of tannins in ruminant feeding.