Objectives To evaluate the effect of lemon essential oil (LEO) on salivary bacteria and volatile sulfur compound (VSC) production of patients with halitosis. Materials and Methods Saliva of five patients with halitosis was collected, after adding different concentrations (0.563-9 mg/ml) of LEO, detecting the growth of salivary bacteria, the formation of biofilm, and VSC production, and compare the difference of different concentrations of LEO on bacterial growth and VSC production. 48 volunteers were randomly divided into 4 groups. After gargling with LEO, cetylpyridinium chloride (CPC), chlorhexidine (CHX), and hydrogen peroxide (H2O2) separately measure changes of VSC production and pH values at 30, 45, 60, 90, and 120 min and then compare the differences at different time points within group. Results Compared with the negative control group, under subinhibitory concentrations of LEO (0.563-2.25 mg/ml), the biofilm formation and VSC production of salivary bacteria in LEO group were significantly inhibited (p < 0.05). Compared with the baseline, the VSC production of subjects decreased after rinsing with the LEO in 60 min (p < 0.05). After gargling with LEO, the pH value rose significantly in 30 min and reverted to the baseline level at 120 min (p < 0.05). Conclusions Lemon essential oil can inhibit the growth of salivary bacteria and reduce VSC production of patients with halitosis.