Six dual-flow continuous culture fermenters were used over a 10-day experimental period to compare the digestibilities of dry matter (DM) and fiber, ruminal fermentation profile, and nitrogen metabolism of rumen microbial populations obtained from an inoculum of chamois (Rupicapra pyrenaica pyrenaica) and cattle (Bos taurus). The aim of this work was to test the fermenters with inoculum from chamois and to compare the function of ruminal microbial population of chamois and cattle under the same conditions of diet and passage rate. Fermenters were fed 56 g DM daily of a diet consisting of 75% Italian ryegrass hay (Lolium multiflorum Lam. var. westerwoldicum) and 25% alfalfa hay (Medicago sativa L.). Both inocula were adapted to the in vitro conditions after 6 days. There were no differences in DM and fiber digestibilities, and total volatile fatty acids concentration (VFA) between cattle and chamois inocula. The proportion of propionate was higher (25.8 vs. 17.9 mol/100 mol), and that of butyrate was lower (9.9 vs. 13.8 mol/100 mol) in chamois inoculum than in cattle inoculum. Ammonia nitrogen concentration was greater (10.6 vs. 6.7 mg/100 ml), but microbial protein synthesis was lower (41.1 vs. 55.3% of total nitrogen flow) in chamois inoculum than in cattle inoculum. These results suggest that microbial populations from chamois and cattle inocula, maintained under the same conditions of diet and passage rate, have a similar degradation capacity, producing the same amounts of VFA, but with a different fermentation profile, and different microbial protein synthesis. The dual-flow continuous culture fermenters could be a useful tool to study ruminal fermentation in wild species such as Pyrenean chamois.