This study aimed to increase the bioactivity of essential oils by fractionation, combination, and emulsification. In this regard, pharmaceutical quality Rosmarinus officinalis L. (rosemary), Salvia sclarea L. (clary sage), Lavandula latifolia Medik. (spike lavender), and Matricaria chamomilla L. (chamomile) essential oils were fractionated by vacuum-column chromatography. The main components of the essential oils were verified, and their fractions were characterized by thin layer chromatography, gas chromatography-flame ionization detector, and gas chromatography/mass spectrometry. Besides, oil-in-water (O/W) emulsions of essential oils and diethyl ether fractions were obtained by the self-emulsification method, followed by droplet size, polydispersity index, and zeta potential value measurements. The in vitro antibacterial effects of the emulsions and binary combinations (10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, v:v) against Staphylococcus aureus were determined by microdilution. In addition, the in vitro anti-biofilm, antioxidant, and anti-inflammatory effects of emulsion formulations were evaluated. According to the experimental results, fractionation and emulsification enhanced essential oil in vitro antibacterial, anti-inflammatory, and antioxidant effects due to increased solubility and nano-sized droplets. Among 22 different emulsion combinations, 1584 test concentrations resulted in 21 cases of synergistic effects. The mechanism of the increase in biological activities was hypothesized to be higher solubility and stability of the essential oil fractions. Food and pharmaceutical industries may benefit from the procedure proposed in this study.