Strains of Staphylococcus aureus have shown a high rate of resistance to conventional antimicrobials; on the other hand, stingless bee honey has shown antimicrobial capacity and may be an alternative product to fight against these infections. There are no reports about the antimicrobial activity of honey from the Melipona bicolor species. In this context, the aim of this study was to test Melipona bicolor honey against strains of Staphylococcus aureus, a strain that is multi-resistant to antimicrobials (Clindamycin, Erythromycin, Streptomycin, Oxacillin, Penicillin, and Vancomycin) and to determine the minimum inhibitory concentration. The strain of Staphylococcus aureus used was isolated from raw milk, collected directly from a dairy farm and stored in a solid nutrient medium at room temperature. The antibacterial potential of M. bicolor honey (pH 3,07) against multidrug-resistant and sensitive S. aureus strains was assessed using the minimum inhibitory concentration (MIC) method in 96-well microdilution plates. From the isolated colonies, the inoculum was prepared with a standard concentration of colony forming units (CFUs) at a concentration of 0.5 McFarland (1.5 X 10⁸ CFU/mL). The dilutions of honey tested were made at concentrations of between 5 and 1000 µl by sterilization through filtration with a 0.22µm 30mm PES membrane syringe filter and the dilution of the filtered aliquot in Muller–Hinton broth (MHB). Then, 50 µl of each dilution tested was added to the wells of 96-well plates, followed by 50 µl of the suspensions of the bacteria tested in isolation. The microplate was then incubated at 35°C for 24 hours. The MIC value was then investigated by applying 30 µl of the viability and aerobic cell metabolism detector dyes Resazurin and 2,3,5-triphenyltetrazolium chloride (TTC). The growth of the strains was observed at all the concentrations tested, so the antimicrobial effect of M. bicolor honey against the S. aureus strains studied was not confirmed.