Piperine is an alkaloid, but its therapeutic efficacy is limited due to poor aqueous solubility. In this study, piperine nanoemulsions were prepared using oleic acid (oil), Cremophore EL (surfactant), and Tween 80 (co-surfactant) using the high-energy ultrasonication approach. The optimal nanoemulsion (N2) was further evaluated using transmission electron microscopy, release, permeation, antibacterial, and cell viability studies based on minimal droplet size and maximum encapsulation efficiency. The prepared nanoemulsions (N1-N6) showed a transmittance of more than 95%, a mean droplet size between 105 ± 4.11 and 250 ± 7.4 nm, a polydispersity index of 0.19 to 0.36, and a ζ potential of -19 to -39 mV. The optimized nanoemulsion (N2) showed significantly improved drug release and permeation compared with pure piperine dispersion. The nanoemulsions were stable in the tested media. The transmission electron microscopy image showed a spherical and dispersed nanoemulsion droplet. The antibacterial and cell line results of piperine nanoemulsions were significantly better than the pure piperine dispersion. The findings suggested that piperine nanoemulsions may be a more advanced nanodrug delivery system than conventional ones.