Unlocking the Multifunctional Properties of <i>Litsea cubeba</i> Essential Oil: Composition, Antimicrobial, Antibiofilm and Insecticidal Activities

ABSTRACT Litsea cubeba essential oil (LCEO) is recognised for its broad spectrum of biological activities, including antimicrobial and insecticidal effects, making it a promising natural alternative to synthetic agents. The aim of this study was to evaluate the chemical composition, antimicrobial in vitro and in situ, antibiofilm and insecticidal activities of LCEO against selected bacterial and yeast strains, as well as against the storage pest Callosobruchus maculatus . The antimicrobial activity was assessed by disc diffusion assay, minimum inhibitory concentrations (MIC₅₀ and MIC₉₀) and in situ vapour‐phase tests on fresh produce (apple, pear, potato, parsley). Antibiofilm effects were determined using the crystal violet method, and MALDI‐TOF MS spectral profiling was applied to compare planktonic and biofilm‐associated Salmonella enterica cells on different surfaces over time. Insecticidal activity was tested in fumigation assays at different concentrations. GC–MS analysis revealed that citral isomers (neral 31.5%, α‐citral 30.1%) and limonene (19.6%) were the dominant constituents of LCEO, accounting for more than 80% of the total volatile fraction. LCEO exhibited significant antimicrobial activity, with Gram‐positive bacteria showing larger inhibition zones (up to 15.67 mm for S. aureus ) compared with Gram‐negative strains and yeasts. MIC values confirmed strong effects against Y. enterocolitica (0.156 mg/mL) and C. albicans (0.077 mg/mL), while E. coli was the most resistant (2.218 mg/mL). In situ assays revealed matrix‐dependent responses: on pears, inhibition exceeded 90% for both Gram‐positive and Gram‐negative bacteria at 500 μg/L, while on potatoes maximum bacterial inhibition was recorded at the lowest concentration (62.5 μg/L), followed by a biphasic decline. Biofilm formation of S. enterica was effectively suppressed (MIC₅₀ = 0.33 mg/mL; MIC₉₀ = 0.35 mg/mL), and MALDI‐TOF MS analyses demonstrated time‐ and surface‐dependent spectral changes between planktonic and biofilm‐associated cells. Insecticidal assays confirmed complete mortality of C. maculatus adults at ≥ 50% LCEO. In conclusion, LCEO displays strong and multifaceted antimicrobial, antibiofilm and insecticidal activities, supporting its potential application as a natural preservative and biocontrol agent.