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Basil (<i>Ocimum basilicum</i>) is a commonly used herb; it also contains essential oils and other valuable compounds. The basil oil obtained has a pleasant aroma, but also a broad spectrum of biological activity. This work reports on the chemical composition, antioxidant, antimicrobial and anti-insect activity in vitro and in situ of <i>Ocimum basilicum</i> essential oil (OBEO) obtained by steam distillation of fresh flowering plants. Gas chromatography–mass spectrometry, DPPH, agar and disc diffusion and vapor phase methods were used to analyze the OBEO properties. The analysis of the chemical composition of OBEO showed that its main components were methyl chavicol (88.6%), 1,8-cineole (4.2%) and α-trans-bergamotene (1.7%). A strong antioxidant effect was demonstrated at the level of 77.3%. The analysis of antimicrobial properties showed that OBEO exerts variable strength of inhibiting activity against various groups of microorganisms. The growth inhibition zones ranged from 9.67 to 15.33 mm in Gram-positive (G⁺) and Gram-negative (G⁻) bacteria and from 5.33 to 7.33 mm in yeast. The lowest measured minimal inhibition concentration (MIC) was 3.21 µL/mL against Gram-negative <i>Azotobacter chrococcum</i> and Gram-positive <i>Micrococcus luteus</i>. The antimicrobial activity of in situ vapor phase of OBEO was also confirmed on apples, pears, potatoes and kohlrabi. The highest insecticidal activity against <i>Pyrrhocoris</i><i>apterus</i>, observed at the concentration of 100%, caused the death of 80% of individuals. Due to its broad spectrum of activity, OBEO seems an ideal candidate for preserving fruit and vegetables.