Background: The aim of this study was to investigate the effect of diets containing organic zinc and a mixture of medicinal herbs on rumen microbial fermentation and histopathology in lambs.Methods: Twenty-eight lambs were divided into four groups: unsupplemented animals (Control), animals supplemented with organic zinc (Zn, 70 mg Zn/kg diet), animals supplemented with a mix of dry medicinal herbs (Herbs, 100 g DM/d) and animals supplemented with both zinc and herbs (Zn+Herbs). Each lamb was fed a basal diet composed of meadow hay (700 g DM/d) and barley (300 g DM/d). Fumaria officinalis L. (FO), Malva sylvestris L. (MS), Artemisia absinthium L. (AA) and Matricaria chamomilla L. (MC) were mixed in equal proportions. The lambs were slaughtered after 70 days of the experiment. The rumen content was used to determine the fermentation parameters in vitro and in vivo and to quantify the microbes by molecular and microscopic methods. Samples of fresh rumen tissue were used for histopathological evaluation.Results: Quantitative analyses of the bioactive compounds identified 3.961, 0.654, 6.482 and 12.084 g/kg DM phenolic acids and 12.211, 6.479, 0.349 and 2.442 g/kg DM flavonoids in FO, MS, AA and MC, respectively. The alkaloids content in FO was 6.015 g/kg DM. The effect of diets on values of total gas, methane and n-butyrate in vitro was observed (P < 0.046 and P < 0.001, respectively). Relative quantification by real-time PCR revealed a decrease in the total rumen bacterial population in the lambs in the Zn and Zn+Herbs groups (P < 0.05). A shift in the relative abundance of Ruminococcus albus, Streptococcus bovis, Butyrivibrio proteoclasticus and R. flavefaciens was observed in the Zn group. Morphological observation showed focal mixed infiltration of inflammatory cells within the lamina propria of the rumen in the Zn+Herbs group. Conclusion: The effect of the organic zinc and the herbal mix on rumen fermentation parameters in vitro was not confirmed in vivo because the rumen microbiota of lambs probably adapted to the zinc-supplemented diets. However, long-term supplementation of a diet combining zinc and medicinal herbs can diminish the health of the ruminal epithelium of lambs.