Pressure-driven membrane separation processes (MSP) are widely used in dairy processing. It is well known that MSP can improve economic profitability of dairy manufacturing by increasing cheese yields or by valorization of by-products such as whey and permeates. However, the contribution of MSP to the environmental impacts of dairy products is not clearly set out. Eco-efficiency is a concept that links both the economic and the environmental dimensions of a system. The objective of this study was to assess the contribution of MSP to the eco-efficiency of Cheddar cheese production in the Quebec province using a validated process simulation software. Three scenarios were compared: a conventional scenario (without MSP), a scenario integrating ultrafiltration (UF) operations at the standardization stage of cheese milk (5.93% proteins, wt/wt), and one final scenario integrating reverse-osmosis and UF operations in the by-product valorization processes (WPC 80 and permeate powder). By including mass balance, life cycle assessment, and net margin calculations in the process simulation, the study allowed for calculations of 4 eco-efficiency indicators. Each indicator expressed the net margin obtained per one environmental damage unit. The environmental damages considered were related to climate change, ecosystem quality, non-renewable resources, and human health. Results showed that even if the introduction of UF at the cheese milk standardization stage allowed for significantly improved productivity (+84%), it did not allow for improvement of the eco-efficiency of the process (8 to 16% less eco-efficient). However, introduction of MSP in the by-product valorization processes were highly beneficial to eco-efficiency since they allowed for reduction of non-renewable energy consumptions (reduced by more than 30% at drying stage) and increase in co-products values (addition of 2 to 5 percentage points to the net margin rate). This led to an overall improvement of the dairy process eco-efficiency of 3 to 24%. The process simulation software, validated using industrial data, allowed for exploration of possibilities and comparison of scenarios without the need for trials.