The advantages of bio-based plasticizers and the differences in their biotoxicity and plasticizing mechanisms compared to phthalate plasticizers have rarely been systematically investigated. Epoxidized soybean oil (ESO), triphenyl phosphate (TCP), and acetyl tributyl citrate (ATBC) were specifically chosen for a rigorous comparative analysis with diocty phthalate (DOP), employing a blend of toxicological studies, characterization methodologies and molecular dynamics (MD) simulations. Based on the blood routine indicators and liver tissue pathology analysis in SD rats, the biocompatibility ranking is as follows: ESO >
ATBC >
TCP >
DOP. When the plasticizer content is 40 wt%, ATBC/PVC and ESO/PVC exhibit superior elongation at break compared to DOP/PVC. MD results indicate that ATBC, ESO, TCP, and DOP can all spontaneously disperse in PVC. Among them, ESO exhibits the highest compatibility with PVC attributed to the interaction forces. For ESO/PVC, interactions include electrostatic forces between polar groups, van der Waals forces, and the entangling of alkyl chains. For ATBC/PVC, the interaction is primarily due to the hydrophobic alkyl chains entangling with PVC through hydrophobic interactions. These observations have been corroborated by MD results, providing additional insights into the underlying microscopic mechanisms. This study offers theoretical support for the broader utilization of environmentally friendly plasticizers.