Biological, and particularly antimicrobial, activities have been demonstrated for the essential oil of propolis samples worlwide, yet their mutagenic effects remain unknown. To correlate antimicrobial effects with mutagenic risks, the present study evaluated the antifungal and antibacterial activities of the essential oil obtained from brown propolis collected from the Cerrado biome in Midwest Brazil (EOP), testing it against nine pathogenic microorganisms. Evaluation of mutagenic potential was based on the somatic mutation and recombination test (SMART) performed on wing cells of standard (ST) and high-bioactivation (HB) crosses of Drosophila melanogaster. EOP was extracted by hydrodistillation, and sesquiterpenes were characterized by GC–MS as its major constituents. The crude oil proved active against Cryptococcus neoformans and Enterococcus faecalis, as did two of its major constituents, spathulenol and (E)-nerolidol – the latter being also active against Staphylococcus aureus – isolated using chromatographic procedures. No significant increase in the number of somatic mutations was observed in the offspring of ST or HB crosses – the latter exhibiting enhanced levels of metabolizing enzymes of the cytochrome P450 type – treated with 0.05%, 0.1%, and 0.2% EOP. These findings revealed no mutagenic activity of EOP, even when tested against the HB strain, and demonstrated that its antimicrobial activities are not associated with DNA damage induction (investigated with SMART), suggesting the potential of EOP as a natural preservative.