Antimicrobial in vitro and in situ efficacies of Cymbopogon citratus essential oil (lemongrass, LGEO) against 17 spoilage microorganisms (bacteria, yeasts and fungi) were evaluated. Additionally, its chemical composition, and antioxidant and antibiofilm activities were investigated. The LGEO exhibited a strong antioxidant activity (84.0 ± 0.1%), and its main constituents were citral (61.5%), geraniol (6.6%) and 1,8-cineole (6.4%). An in vitro antimicrobial evaluation revealed the lowest inhibition zone (1.00 ± 0.00 mm) in Pseudomonas fluorescens, and the highest inhibition zone (18.00 ± 2.46 mm) in Candida krusei. The values for the minimal inhibitory concentration were determined to be the lowest for Salmonella enteritidis and the highest for C. albicans. Furthermore, the concentration of ≥250 µL/L of LGEO suppressed the growth of Penicillium aurantiogriseum, Penicillium expansum, Penicillium chrysogenum and Penicillium italicum. The changes in the molecular structure of the biofilms produced by Pseudomonas fluorescens and Salmonella enteritidis, after their treatment with LGEO, confirmed its action on both biofilm-forming bacteria. Moreover, an in situ antimicrobial activity evaluation displayed the most effective inhibitory effectiveness of LGEO against Micrococcus luteus, Serratia marcescens (250 µL/L) and Penicillium expansum (125, 250 and 500 µL/L) growing on a carrot. Our results suggest that LGEO, as a promising natural antimicrobial agent, can be applied in the innovative packaging of bakery products and different types of vegetables, which combines commonly used packing materials with the addition of LGEO.