This study investigated the hydrogen mass transfer limitations in a biotrickling filter inoculated with hydrogenotrophic methanogens for biogas upgrading. A highly sensitive dissolved hydrogen probe allowed measuring concentrations in real-time. Experiments were conducted to test the mass transfer resistance in the gas and liquid films. Results demonstrated that the main resistance resides in the trickling liquid film and that promoting direct gas-biofilm mass transfer could improve upgrading performance by about 20%. Increasing the gas velocity (keeping a constant gas contact time) lowered the upgrading capacity. This was explained by the lowering of the concentration to the average concentration throughout the bed, which resulted a lower reaction rate. At extended gas contact times, the bioreactor shifted from microbial to diffusion limitation, causing lower upgrading capacities. Methane-containing biogas mimics (H/CH/CO) were successfully upgraded to natural gas pipeline standards (>97% methane) with only minor performance reduction compared to upgrading just a H/CO mixture.