Hydroalcoholic propolis extracts from the bee species Melipona quadrifasciata have been shown to possess antimicrobial activity against different mollicute strains, but a methanolic extract (ME) could contain an increased diversity of nonpolar bioactive components with a potentially higher antimicrobial activity. The ME obtained by maceration of the propolis sample was fractionated with solvents of different polarities and then, purified by silica gel column chromatography through biomonitoring of its antimicrobial activity against mollicute strains. Analysis by gas chromatography-mass spectrometry (GC/MS) enabled the identification of compounds using the NIST library. Minimum inhibitory concentrations (MICs) of the samples were determined by broth microdilution. Anti-adhesive assays were performed with Mycoplasma pneumoniae cells. The hexane (MIC=62.5 mg/L) and dichloromethane (MIC=125 mg/L) fractions presented the most promising results against M. pneumoniae. They were fractionated into 74 subfractions, and even the best ones did not show better results (MIC>250 mg/L) than their original fractions, likely due to the loss of terpene compounds that seem to act in synergy. The dichloromethane subfraction FD4 was highlighted in the anti-adhesive assay with an inhibitory activity of 21.6 %. A synergistic effect of the nonpolar compounds in M. quadrifasciata propolis may be responsible for its antibacterial activity, but several purified components can improve its anti-adhesive properties.