The growing emphasis on food safety and healthier lifestyles, driven by industrial expansion and scientific priorities, has highlighted the necessity of managing harmful microorganisms to guarantee food quality. A significant challenge in this domain is the control of pathogens that are capable of forming biofilms, entering a sessile state that enhances their resistance to broad-spectrum antibiotics. Essential oils, renowned for their antibacterial properties, present a promising natural alternative for food preservation. In this study, we analyzed the chemical composition of Santalum album essential oil (SAEO) using GC-MS, identifying (Z)-α-santalol (57.1%) as the primary constituent. Antimicrobial activity was confirmed through disc diffusion and minimum inhibitory concentration (MIC) assays against Gram-positive and Gram-negative bacteria and yeast from the genus Candida. Additionally, in situ experiments demonstrated that vapor-phase SAEO effectively inhibited Serratia marcescens on the food model, supporting its potential as a natural preservative. MBIC assays, crystal violet staining, and MALDI-TOF MS analysis on S. enterica biofilms were used to further evaluate the antibiofilm effects of SAEO. The crystal violet assay revealed a strong antibiofilm effect, while the MALDI-TOF MS analysis showed changes in the bacterial protein profiles on both glass and plastic surfaces. SAEO also showed significant anti-Salmonella activity on vacuum-packed carrot slices. SAEO outperformed the control samples. The insecticidal activity against Megabruchidius dorsalis was also studied in this work, and the best insecticidal activity was found at the highest concentrations. These findings indicate that SAEO could serve as a valuable component in food preservation, with notable antibacterial and antibiofilm benefits.