Pinus represents a potential source of valuable bioactive substances. In this study, the chemical composition and biological activity of essential oils extracted from Pinus mugo Turra needles (PMEO) were investigated. GC/MS analyses were used to assess the volatile elements found in the PMEO sample. The antioxidant activity was evaluated using DPPH and ABTS techniques. Antimicrobial properties were evaluated against yeasts and bacteria (both Gram-positive and Gram-negative) using the disc diffusion method, minimal inhibition concentration and vapour phase on fruit and vegetable models. Mass spectrometry and the crystal violet technique were used to measure the antibiofilm activity. The contact application method was used to assess the insecticidal activity of different concentration of PMEO against Harmonia axyridis. The GC/MS analysis identified β-phellandrene (20.5%), α-pinene (19.4%), δ-3-carene (16.7%), β-pinene (11.1%) and β-myrcene (9.4%) as main constituents. PMEO exhibited antioxidant activity against DPPH and ABTS radical species, with IC50 values of 1.92 and 1.14 mg/mL, respectively. PMEO showed an inhibitory effect against microbial strains ranging from 6.67 to 13.33 mm. By employing the microdilution method, PMEO showed antimicrobial activity with MIC50 values ranging from 2.52 to 7.41 mg/mL and MIC90 values ranging from 2.72 to 7.7 mg/mL. The in situ antimicrobial investigations revealed that PMEO exerts the highest activity against E. aerogenes and P. putida on apple and beetroot models. Furthermore, PMEO exhibited noteworthy potential against biofilm-forming S. enterica growing on plastic and glass surfaces. Additionally, an evaluation of the PMEO insecticidal potential was performed using H. axyridis. Insecticidal properties against H. axyridis were observed at concentrations of 50% and 100%. PMEO displayed good antioxidant, antibacterial and insecticidal properties, significant antibiofilm features and excellent preservative properties for extending the shelf life of foods.