The increasing concerns about pollution, scarcity of resources, and the excessive consumption of petrochemical-based materials have created a need for developing eco-friendly alternatives in separation technologies. Among these, plant-derived and agricultural waste-based biopolymers have become promising materials as they are biodegradable, reusable, non-toxic, and functionally versatile. Water purification, gas separation, and industrial effluent are examples of the separation technologies currently benefiting from natural biopolymers. This chapter thoroughly examines the integration of natural polymers in different separation technologies. Currently, in adsorption, membrane filtration, pervaporation, and flocculation, plant- and agricultural waste-derived biopolymers, like starch, alginate, cellulose, lignin, carrageenan, and guar gum, are being considered as alternatives. These biopolymers are used for the purification of wastewater, adsorption of heavy metals, CO2 capture, and oil and water separation. Biodegradability, lifecycle impacts, and environmental impact assessment (EIA) of biopolymers will be addressed. Furthermore, the current limitations and challenges in the application of biopolymers in separation technologies, such as their scalability and mechanical durability, must be improved to meet the synthetic polymer standards. Future perspectives include bridging the gap between research and industry, promoting industrial innovation, and environmental protection.