Hydrogenases are efficient biocatalysts for H2 production and oxidation with various potential biotechnological applications.[NiFe]-class hydrogenases are highly active in both production and oxidation processes—albeit primarily biased to the latter—but suffer from being sensitive to O2.[NiFeSe] hydrogenases are a subclass of [NiFe] hydrogenases with, usually, an increased insensitivity to aerobic environments. In this study we aim to understand the structural causes of the low sensitivity of a [NiFeSe]-hydrogenase, when compared with a [NiFe] class enzyme, by studying the diffusion of O2. To unravel the differences between the two enzymes, we used computational methods comprising Molecular Dynamics simulations with explicit O2 and Implicit Ligand Sampling methodologies. With the latter, we were able to map the free energy landscapes for O2 permeation in both enzymes. We derived pathways from these energy landscapes and selected the kinetically more relevant ones with reactive flux analysis using transition path theory. These studies evidence the existence of quite different pathways in both enzymes and predict a lower permeation efficiency for O2 in the case of the [NiFeSe]-hydrogenase when compared with the [NiFe] enzyme. These differences can explain the experimentally observed lower inhibition by O2 on [NiFeSe]-hydrogenases, when compared with [NiFe]-hydrogenases. A comprehensive map of the residues lining the most important O2 pathways in both enzymes is also presented.