The antibacterial potential of the most diverse medicinal plants has benefited humanity for centuries, and precisely because of this, the number of studies investigating the antimicrobial activity of essential oils and their components is increasing. However, the hydrophobic character of the essential oils has made the experiments difficult, requiring the use of organic solvents in the tests in order to avoid such complications. Among the most commonly used solvents are dimethylsulfoxide (DMSO / C₂H₆OS). To date, the literature has not yet determined a standardization of the usual concentration of DMSO suitable for bacterial experiments, so that its use does not check the efficacy of the tested phytoconstituent by interactions between the solvent and the exploited compound. In view of this reality, the present study intends to standardize the DMSO concentrations that do not interfere in the viability of the bacterial strains of Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 25922 and Enterococcus faecalis ATCC 29212. In order to reach this objective, the disc diffusion method in Müller Hinton Agar of a reagent impregnated on a filter paper disc was used, using different concentrations of DMSO to determine the minimum inhibitory concentration (MIC) of the five microorganisms listed. The experiment was carried out in triplicate in order to safeguard the effectiveness of the method tested. The incubation was done in an oven at 35º C, for a period of 24 hours. For the test method the conclusions of the tests performed were expressed by the arithmetic mean of the diameter of the inhibition halos formed around the disks. The test demonstrates that for the concentrations and microorganisms tested, DMSO provides safety in its use.