Several in vitro fermentation studies had demonstrated the efficacy of Mootral, a patent-pending natural feed supplement, in reducing methane gas production. In order to test the supplement's application in a commercial farm, Jersey (n = 121) and Holstein-Friesian (HF, n = 275) lactating cows received 500 g of pellets containing 3% Mootral powder for 12 weeks. Their enteric methane gas production was monitored using a hand-held laser methane detector. During 7 weeks of pre-Mootral supplementation period, dry matter intake (DMI), milk yield and milk quality (fat, protein, urea, pH, bulk tank somatic cell count (BTSCC)) were monitored for both herds and continued during 12 weeks of Mootral supplementation (Mootral period), and 4 weeks after withdrawal of Mootral (post-Mootral period). Feed samples were analysed during each period, and feed efficiencies (FE) for each herd were calculated. Compared to the baseline, the methane gas produced by the Jersey and HF cows during the Mootral period were lower by 38.3% and 20.7% (p < 0.05), respectively. DMI for Jerseys were greatest during the pre-Mootral period (p < 0.05), while no difference was recorded for the HF cows. Milk yield increased significantly (p < 0.05) for both herds during Mootral supplementation. FE increased significantly for the Jersey herd during the Mootral and post-Mootral periods (p < 0.05). Statistically significant changes were detected for urea in both herds, and BTSCC for HF cows, but these did not adversely affect milk quality. Results demonstrated Mootral reduces enteric methane production in lactating cows while increasing milk yield without affecting milk quality.