Background: Microbial resistance to antibiotics is a global public health problem, which requires urgent attention. Platonia insignis is a native species from the eastern Brazilian Amazon, used in the treatment of burns and wounds. Objectives: To evaluate the antimicrobial activity of the hydroalcoholic extract of P. insignis (PIHA), the ethyl acetate fraction (PIAE), and its subfraction containing a mixture of biflavonoids (BF). Moreover, the effect of these natural products on the antibiotic activity against S. aureus strains overexpressing efflux pump genes was also evaluated. Methods: Minimal inhibitory concentrations were determined against different species of microorganisms. To evaluate the modulatory effect on the Norfloxacin-resistance, the MIC of this antibiotic was determined in the absence and presence of the natural products at subinhibitory concentrations. Inhibition of the EtBr efflux assays were conducted in the absence or presence of natural products. Results: PIHA showed a microbicidal effect against S. aureus and C. albicans, while PIAE was bacteriostatic for S. aureus. PIAE and BF at subinhibitory concentrations were able to reduce the MIC of Norfloxacin acting as modulating agents. BF was able to inhibit the efflux of EtBr efflux in S. aureus strains overexpressing specific efflux pump genes. Conclusion: P. inignisis, a source of efflux pump inhibitors, including volkensiflavone and morelloflavone, which were able to potentiate the Norfloxacin activity by NorA inhibition, being also able to inhibit QacA/B, TetK and MsrA. Volkensiflavone and morelloflavone could be used as an adjuvant in the antibiotic therapy of multidrug resistant S. aureus strains overexpressing efflux pumps.