Ruminal methanogenesis is considered an inefficient process as it can result in the loss of 4 to 12% of the total energy consumed by the ruminant. Recent studies have shown that compounds such as nitroethane, 2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-1-propionic acid are capable of inhibiting methane production during in vitro studies. However, all of these nitrocompounds came from a synthetic origin, which could limit their use. In contrast, some plants of the Astragallus genus produce a natural nitrocompound, although its anti-methanogenic effect has not been evaluated. To determine the anti-methanogenic effect, in vitro cultures of freshly collected mixed populations of ruminal microbes were supplemented with A. mollissimus extracts (MISER). Cultures supplemented with 2-nitroethanol, ethyl 2-nitroacetate, or nitroethane were used as positive controls whereas distilled water was added to the untreated control tubes. After a 24 h incubation period, the methane production was reduced by more than 98% for the samples treated with A. mollissimus extract (P < 0.05) compared to the untreated controls (10.2 ± 0.1 mmol mL−1 incubated liquid). Cultures supplemented with MISER produced a greater (P < 0.05) amount of total VFA, compared to the rest of treated and untreated cultures. Considering that there are significant differences between MISER treatment, positive controls and untreated cultures (P < 0.05) regarding the amounts of total gas, gas composition (CH4 and H2), and the amount of VFA produced, it is concluded that Astragallus mollissimus poses an alternative strategy to reduce ruminal methanogenesis. To further explore such alternative, it is necessary to determine if the metabolization byproducts are safe and/or useful for the animal.