Pseudomonas aeruginosa and Staphylococcus aureus are highly resistant microorganisms that contribute to prolonged hospital stays and increased mortality. Developing new antimicrobial agents is essential to address this global health challenge. Nanoemulsions (NE) containing essential oils (EOs) and phenolic compounds with antimicrobial activity represent a promising alternative. This study reports, for the first time, the formulation of a NE containing Cymbopogon flexuosus and eugenol (NECE) and its antimicrobial activity against P. aeruginosa and S. aureus. NECE exhibited suitable physicochemical properties (mean size < 200 nm, PDI < 0.3, and negative zeta potential) and remained stable for 90 days at 4 °C while maintaining antimicrobial activity. It showed bactericidal effects at 2.5 mg/mL against P. aeruginosa and 0.625 mg/mL against S. aureus. Moreover, NECE improved the biocompatibility of the free oil (FO) in Peripheral Blood Mononuclear Cells (PBMCs). Altogether, these findings demonstrate, for the first time, that NECE is a stable nanoemulsion with enhanced antimicrobial activity and biocompatibility, supporting its potential as a safe and effective topical strategy against wound-associated pathogens.