Plant-derived essential oils are volatile hydrophobic compounds with significant antimicrobial activities. Considering the rise of antimicrobial resistance, these natural products have been highlighted as efficient weapons against multidrug-resistant microorganisms. This work aimed to investigate the phytochemical composition and antimicrobial activity of Piper regnellii essential oil against strains of Escherichia coli, Staphylococcus aureus, Candida albicans, and Candida tropicalis. Phytochemical analysis was performed through gas chromatography coupled with mass spectrometry (CG/MS). The intrinsic antimicrobial activity and the ability of the essential oil to modulate antimicrobial resistance were assessed using the broth microdilution method. Fungal virulence inhibition was analyzed by measuring the growth of hyphae in microculture chambers. Phytochemical characterization revealed a predominance of phenylpropanoids, including apiol (70.79%) and dilapiol (15.05%) as major constituents. While presenting clinically ineffective antibacterial effects (MIC 1.024 mg/mL for all strains), the essential oil potentiated the activity of gentamicin against E. coli at concentrations above 20 mg/mL. Piper regnellii essential oil showed clinically ineffective antifungal activity with IC50 values above 500 mg/mL. However, it was found to potentiate the activity of fluconazole against C. tropicalis at concentrations ranging from 32 mg/mL to 1024 mg/mL. Furthermore, the morphological transition was inhibited by culturing C. albicans and C. tropicalis with different concentrations of the essential oil. Together, our results indicate that P. regnellii essential oil presents promising antifungal effects. However, the mechanisms underlying its interference on Candida virulence remain to be further investigated.