In this study, we evaluated propolis as a safe alternative for ruminants feeding free of antibiotic additives and growth promoters. Phenolic compounds with antibiotic activity mainly against gram-positive bacteria, in the same manner as ionophores, are present in propolis. We evaluated the effects of different levels of ethanolic extract of propolis (EEP) at 30% on the in vitro production of gases by bacteria. Feed was formulated to contain 50% ground elephant grass (Pennisetum purpureum) and 50% concentrate, consisting of corn, soybean meal and core minerals and vitamins. EEP was added to the concentrate at 0, 6, 12, 24, and 36 mL/kg. A sample of 1000mg was weighed in nylon bags, with a weight of 20 mg/cm², sealed and added to the fermentation vials (160mL) previously injected with CO2, with 4 replicates per treatment. To each vial was added 90mL of buffer solution and 10 mL of ruminal fluid spray kept under CO₂. The vials were sealed, placed in a room at 39°C and stirred every 2 h. Gas pressure readings were made at 2, 4, 6, 8, 9, 11, 12, 14, 17, 20, 24, 28, 34, 48, 72, 96, and 120 h after incubation. Statistical analyzes were performed using SAS 9.1 (SAS Institute Inc., Cary, NC). Data were previously checked for normality of residuals by Shapiro-Wilk test (PROC UNIVARIATE) and the variances compared by orthogonal contrasts (PROC GLM) with significance level of 5%. When significant, the regression equations were determined (PROC MIXED). As EEP levels were not equidistant, it was generated vectors of each contrast using PROC IML. There were no effects (P > 0.05) of EEP levels to the fibrous (0.0715 ± 0.02 mL/hour), non-fibrous (0.013 ± 0.01 mL/hour), and total (0.083 ± 0.02 mL/hour) carbohydrate fermentation rates. For the potential production of total carbohydrate gases (Vt) there was a quadratic effect (P < 0.05). The lowest value of gas output (77.69 mL/g of DM) was obtained for the inclusion of 23 mL of EEP /kg of concentrate. For the gas production from non-fibrous carbohydrates, the lowest value (63.12 mL/g of DM) was obtained for the inclusion of 24.78 mL of EEP /kg of concentrate. The results suggests that EEP was efficient at inhibiting total gas production by ruminal microorganism in vitro.