Two trials were conducted to investigate the effects of disodium fumarate (DSF; 0.00, 8.00, 10.00 and 12.00 mM) and thyme essential oil (TEO; 0.00, 100.00, 200.00, 300.00 and 400.00 μL L-1) solely and simultaneously (10.00 mM DSF along with 100.00, 200.00, 300.00 and 400 μL L-1 TEO) on in vitro ruminal fermentation of a 50:50 alfalfa hay to concentrate diet. The DSF and TEO did not affect crude protein disappearance, gas production, microbial crude protein synthesis and hydrogen recovery. The DSF addition linearly increased partitioning factor (PF) and molar proportion of propionate and decreased acetate: propionate ratio and methane production. Moreover, 100.00 μL L-1 of TEO decreased ammonia nitrogen, total volatile fatty acids concentration and methane production and increased PF compared to the control. Results of the present study demonstrated that simultaneous use of DSF and TEO can cause a further decrease in methane production and linearly increase in the molar proportion of propionate and efficiency of feed use compared to DSF and TEO solely.