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Agarose is a natural polymer that can form moldable films with unique characteristics that make it a great candidate as an alternate renewable material. Its special characteristics include excellent biocompatibility, the ability to form thermo-reversible films, and a high degree of flexibility when wet. However, agarose films lose much of their flexibility at low moisture content. To assuage this issue, plasticizing agents can be added. A plasticizer is a material that softens another material by disrupting hydrogen bonds and thus improves film flexibility and durability. In this study, four plasticizers (i.e., sucrose, urea, glucose, and glycerol) were chosen and administered to agarose films in varying concentrations and combinations to study the effect different molecules have on agarose’s intermolecular interactions and film properties. To understand the physicochemical, structural, thermal, mechanical, and morphological changes of agarose-based films with various plasticizers, various analytical techniques were implemented including Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), scanning electron microscopy (SEM), X-ray Scattering, and tensile strength. The results indicate that the four plasticizers affected the mechanical and thermal properties, but not the morphological properties of agarose films to varying degrees due to differences in their intermolecular interactions caused by changes in hydrogen bonding groups. This hydrogen bonding effect caused by the differences in plasticizers provides an avenue to precisely tune the properties of agarose films.