Eugenol (EGL) and 2-methylpyrazine (2-MTP) are common food additives that are used as flavors in a variety of foods. To study the effect of food additives on human serum albumin (HSA), it is necessary to deeply study the interaction of EGL and 2-MTP with HSA. Fluorescence, UV–vis absorption, circular dichroism (CD) and computational techniques showed EGL and 2-MTP bind statically to HSA, inducing conformational changes of HSA. We calculated the quenching constants (ksv), binding constants (kq), binding distances (r) and thermodynamic constants (ΔH0, ΔS0, ΔG0) at four different temperatures. Based on thermodynamic analysis and molecular dynamics (MD) simulations, the main forces of 2-MTP-HSA and EGL-HSA systems are hydrophobic interaction and π-π accumulation, respectively. In addition, the interaction alters the physiological activity of HSA, esterase activity and free radical scavenging ability. This study provides valuable insights into the molecular interactions of food additive compounds EGL and 2-MTP with HSA, the findings provide theoretical reference for the development of new food additives and drugs in the future.