Boswellia carterii, known as frankincense, is a fragrant medicinal plant. The essential oil from this plant is often used in traditional medicine or aromatherapy. Due to its positive properties, it has potential applications as an antimicrobial agent in medicine and the food industry. The aim of this study was to evaluate the antimicrobial, anti-insect, and antioxidant activities of frankincense essential oil (FEO). The composition of volatile compounds was determined by GC/MS, and the main components were found to be α-pinene (37.0%), α-limonene (19.8%), and p-cymene (6.3%). The antioxidant activity was evaluated with DPPH and ABTS methods with the resulting inhibition of 73.88 ± 0.35% of DPPH radical (6.27 ± 0.17 TEAC) and 97.09 ± 0.48% of ABTS radical cation (5.21 ± 0.07 TEAC). The antimicrobial activity was the strongest against Candida species with inhibition zones in the range of 38.00–44.00 mm and MIC 50 and MIC 90 values of 11.72 and 12.58 μL/mL, respectively. The inhibition activity of the vapor phase was the highest against G⁺ and G⁻ bacteria growing on a carrot with inhibition ranging from 65.14 to 93.67%. The anti-insect activity against O. lavaterae was determined as 100% at 100% concentration and 50% at 25% concentration. The degradation of biofilm was tested with MALDI-TOF MS, and changes in the protein profile were observed.