The antibacterial efficacy of the methanol extract of the aerial parts of Seseli libanotis by using disc diffusion assay against 107 strains from 52 bacterial species and the chemical composition of the essential oil using GC/MS analysis were studied for the first time. The minimum inhibitory concentrations (MIC) of single compounds were determined by the microbroth dilution method. Gas chromatography–mass spectrophotometry analyses allowed 13 compounds to be determined; the main constituents of the essential oil of aerial parts of Seseli libanotis were trans-caryophyllene (20.39%), spathulenol (11.89%), (−)-caryophyllene oxide (11.47%), euasarone (10.66%) and delta-cadinene (9.16%). The methanol extract of Seseli libanotis had a broad-spectrum antibacterial activity (>14 mm inhibition zone in diameter) in particular against Bacillus cereus, Bacillus dipsauri, Bacillus lentimorbus, Bacillus sphaericus, Bacillus subtilis, Corynebacterium ammoniagenes, Kocuria rosea, Neisseria subflava and Micrococcus lylae. These inhibitory effects are interesting in relation to the prevention of microbial contamination in foods.