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Nanoparticles are materials whose size is less than 100 nm. Because of their distinctive physical and chemical characteristics, nanoparticles have drawn considerable interest in a variety of fields. Biosynthesis of nanoparticles is a green and environmentally friendly technology, which requires fewer chemical reagents, precursors, and catalysts. There are various types of nanomaterials, out of which trimetallic nanoparticles are receiving considerable interest in recent years. Trimetallic nanoparticles possess unique catalytic, biomedical, antimicrobial, active food packaging, and sensing applications as compared to monometallic or bimetallic nanoparticles. Trimetallic nanoparticles are currently synthesized by various methods such as chemical reduction, microwave-assisted, thermal, precipitation, and so on. However, most of these chemical and physical methods are expensive and toxic to the environment. Biological synthesis is one of the promising methods, which includes the use of bacteria, plants, fungi, algae, waste biomass, etc., as reducing agents. Secondary metabolites present in the biological agents act as capping and reducing agents. Green trimetallic nanoparticles can be used for different applications such as anticancer, antibacterial, antifungal, catalytic activity, etc. This review provides an overview of the synthesis of trimetallic nanoparticles using biological agents, and their applications in different areas such as anticancer, antimicrobial activity, drug delivery, catalytic activity, etc. Finally, current challenges, future prospects, and conclusions are highlighted.