The essential oil of Jasminum grandiflorum has very good biological activity. The present study aimed to analyze the chemical composition and biological activity of J. grandiflorum. The main constituents of J. grandiflorum essential oil were benzyl acetate (37%), benzyl benzoate (34.7%) and linalool (9.6%). The antioxidant activity was 58.47%, which corresponds to 220.93 TEAC. The antimicrobial activity was weak to moderate, with inhibition zones ranging from 2.33 to 5.33 mm. The lowest MIC value was against Candida glabrata. The antimicrobial activity of the vapor phase of the essential oil was significantly stronger than that of the contact application. Biofilm analysis using a MALDI-TOF MS Biotyper showed changes in the protein profile of Pseudomonas fluorescens that confirmed the inhibitory effect of J. grandiflorum. The insecticidal potential of the essential oil against Oxycarenus lavatera and Brassicogethes aeneus was also demonstrated. Due to the properties of J. grandiflorum essential oil, it could find application as a biofilm control agent for the shelf-life extension and storage of fruits and vegetables and as a possible insecticidal agent.