Transmembrane β-barrel proteins are key systems for transport phenomena in biology. Based on their broad substrate specificity, they represent good candidates for present and future technological applications, such as DNA/RNA and protein sequencing, sensing of biomedical analytes, and production of blue energy. For a better understanding of the process at the molecular level, we applied parallel tempering simulations in the WTE ensemble to compare two β-barrel porins from Escherichia coli, OmpF and OmpC. Our analysis showed a different behavior of the two highly homologous porins, where subtle amino acid substitutions can modulate critical properties of mass transport. Interestingly, the differences can be mapped to the respective environmental conditions under which the two porins are expressed. Apart from reporting on the advantages of the enhanced sampling methods in assessing the molecular properties of nanopores, our comparative analysis provided new and key results to better understand biological function and technical applications. Eventually, we showed how results from molecular simulations align well with experimental single-channel measurements, thus demonstrating the mature evolution of numerical methodologies for predicting properties in this field crucial for future biomedical applications.