Literature contains limited data on the use of lemon balm in patients infected with the human immunodeficiency virus (HIV). However, data from clinical studies indicate the benefits of this plant’s use in carbohydrate and lipid metabolism disorders. The aim of this study was to examine the effect of lemon balm when used alongside antiretroviral therapy containing efavirenz, darunavir or raltegravir, as well as to determine the positive effects of the use of lemon balm in metabolic syndrome regulation. Both a preliminary and a detailed chemical analysis of lemon balm leaf extract were performed. The antihyperglycemic activity of the extract was investigated in in vitro Ξ±-amylase and Ξ±-glucosidase enzyme inhibition assays. Fermentation of the lemon balm extract, as well as of pure rosmarinic acid with probiotic microorganisms, was also performed in vitro. The investigation performed on patients included patients of the Clinic for Infectious Diseases of the Clinical Center of Vojvodina infected with HIV and with a co-occurring metabolic syndrome. The patients were divided into 3 groups based on the medication they received in antiretroviral therapy, and in addition to the prescribed therapy, they received lemon balm in a dose of 600 mg for 12 weeks. At the beginning and at the end of the study period, the following parameters were measured in patients: the concentration of rosmarinic acid in the patients’ blood, antiretroviral drug concentration (efavirenz, darunavir or raltegravir) in the patients’ blood, the number of copies of HIV in the patients’ blood, hematological, immunological and biochemical parameters, oxidative stress, the activity of enzymes of antioxidant protection, and anthropometric parameters. The tested extract was characterized as a good source of phenolic compounds, with rosemary acid being the most abundant compound. The extract showed in vitro inhibition of both enzymes at relatively low concentrations, with better activity in Ξ±-glucosidase inhibition. In in vitro fermentation, the extract has been shown to undergo intensive metabolism by probiotic strains; rosmarinic acid in the extract was more stable compared to the pure chemical compound. Blood analysis has shown that rosmarinic acid is a marker of the use of lemon balm in plasma, however, this compound is found in the form of a sulfoglucuronide in the blood after 12 weeks of use. No statistically significant differences were found between the concentrations of the tested drugs before and after 12 weeks of investigation. There was no increase in the number of copies of the virus in the blood. The monitored hematological and immunological parameters did not show a statistically significant change in values. Within the monitored biochemical parameters, there were no statistically significant changes that can be associated with the use of lemon balm, with the exception of the studied group of efavirenz, where there was a statistically significant increase in alanine aminotransferases and aspartate aminotransferases, but within reference values. It was shown that after 12 weeks of application of lemon balm, there was a statistically significant reduction in oxidative stress parameters in the studied groups of efavirenz and darunavir; in addition, a statistically significant increase in glutathione-S-transferase activity was shown in the efavirenz study group. Regarding the parameters of anthropometry, an increase in body weight was shown in the examined group of efavirenz after 12 weeks of application of lemon balm. The research presented here shows that the simultaneous use of lemon balm with antiretroviral therapy that includes one of the tested drugs does not affect the concentrations of these drugs. The monitored parameters in the patients showed that the use of lemon balm for 12 weeks in a dose of 600 mg was safe for patients. The presented positive results indicate a beneficial effect on HIV-infected patients with metabolic syndrome.