**Background:** Essential oils (EOs) are complex mixtures of biologically active compounds with potential antimicrobial, antioxidant, and other therapeutic properties. Cupressus sempervirens L. (Cupressaceae) is an evergreen tree used in traditional medicine for respiratory conditions, inflammation, and infections. Despite known biological activities, there were no previous reports on CSEO's antimicrobial efficiency in food preservation using an in situ assay, and literature on its antibiofilm and antiproliferative effects was lacking. This study aimed to comprehensively evaluate the chemical composition, antioxidant, antimicrobial, antibiofilm, insecticidal, and antiproliferative activities of CSEO.
**Methods:** CSEO was purchased from Hanus s.r.o. (Slovakia). Chemical composition was determined by GC/MS and GC-FID using an HP-5MS column. Antioxidant activity was assessed via DPPH and ABTS radical scavenging assays, with results expressed as % inhibition and TEAC values compared to Trolox. Antimicrobial activity was evaluated using disc diffusion (measuring inhibition zone radii from disc edge) and minimum inhibitory concentration (MIC50/MIC90) assays against 3 Gram-negative bacteria (Pseudomonas aeruginosa, Yersinia enterocolitica, Salmonella enterica), 3 Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis), 4 Candida species, and 3 filamentous fungi (Aspergillus flavus, Botrytis cinerea, Penicillium citrinum). An in situ vapor-phase assay was performed on kohlrabi slices as a food model. Antibiofilm activity against Salmonella enterica biofilm on stainless steel and plastic surfaces was analyzed using MALDI-TOF MS Biotyper on days 3, 5, 7, 9, 12, and 14. Insecticidal activity was tested against Oxycarenus lavaterae at concentrations of 3.125–100%. Antiproliferative activity was evaluated by MTT assay on MDA-MB-231 (breast cancer), HCT-116 (colon cancer), JEG-3 (choriocarcinoma), K562 (leukemia), and MRC-5 (normal lung fibroblast) cell lines at 1–200 µg/mL for 24h and 72h.
**Key Results:** Thirty-nine compounds were identified (98.9% of total), with monoterpene hydrocarbons dominating (90.7%). Major compounds were α-pinene (40.5%) and δ-3-carene (24.4%). CSEO showed strong antioxidant activity: 10 µL neutralized 76.32 ± 0.43% of DPPH radical (6.46 ± 0.18 TEAC) and 91.53 ± 0.16% of ABTS radical cation (4.92 ± 0.06 TEAC), with IC50 values of 4.39 ± 0.13 mg/L (DPPH) and 2.96 ± 0.01 mg/L (ABTS). In disc diffusion, strongest antimicrobial effects were against B. subtilis (10.33 ± 1.53 mm) and C. albicans (11.33 ± 1.15 mm). MIC50 values ranged from 40.92 µL/mL (S. enterica) to 374.02 µL/mL (B. subtilis, C. glabrata). In situ vapor-phase assay showed highest inhibition at lower concentrations for most organisms, with S. enterica achieving 84.82 ± 3.00% inhibition at 62.5 µL/mL, though biofilm-producing S. enterica showed growth stimulation at higher concentrations. MALDI-TOF MS analysis demonstrated significant disruption of S. enterica biofilm homeostasis from day 7 onward, with most pronounced effects on days 12 and 14. Insecticidal activity was 100% at full concentration, with LC50 of 21.07% and LC90 of 78.21%. In cell viability assays, CSEO showed time- and dose-dependent antiproliferative effects. K562 cells were most sensitive (IC50 105.06 µg/mL at 24h, 66.56 µg/mL at 72h; viability 47.07% at 200 µg/mL). MRC-5 normal fibroblasts maintained >80.25% viability at 200 µg/mL, indicating acceptable biocompatibility.
**Clinical Implications:** CSEO demonstrates multifaceted biological activities suggesting potential applications in food preservation (particularly vapor-phase antimicrobial application), agricultural pest control (insecticidal activity), and as an antibiofilm agent against Salmonella enterica. The selective antiproliferative effect against cancer cells, especially K562 leukemia cells, with minimal toxicity to normal fibroblasts, warrants further investigation for potential oncological applications. However, these are preclinical findings requiring additional in vivo studies and clinical validation before therapeutic use. The study is limited by its in vitro nature, use of a single commercial oil source, and lack of mechanistic investigations.