Emulsion-based delivery systems have been fabricated and developed to increase the bioavailability of astaxanthin and alpha-tocopherol as active compounds for various biomedical applications. Astaxanthin-alpha tocopherol nanoemulsion (ATNE) is well known for its potential 6.-6.30 effect. The current study investigated ATNE by spontaneous (SENE) and ultrasonication emulsification (USNE) methods to optimally fabricate oil/water nanoemulsion characterized for biomedical applications. The two methods were compared by using a response surface method of 3-level Box-Behnken design (BBD) with significant factors. Transmission electron microscopy (TEM) confirmed spherical-shaped nanoemulsion from SENE and USNE methods and dynamic light scattering (DLS) proved the good stability of the fabricated nanoemulsion. Cytotoxicity studies on three different cancer cells confirmed that the nanoemulsion at higher concentrations was more toxic than one at lower concentrations by accompanying a significant decrease in the cellular viability after 24 and 48 h of exposure. The wound-healing potential using scratch assay evidenced faster healing effect of the nanoemulsion. Both minimal inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) methods confirmed significant antibacterial activity to disrupt the integrity of the bacterial cell membrane. The current results suggested that ATNE act as effectively targeted drug delivery vehicles in the future for cancer treatment applications due to its significant results of anticancer, wound healing, and antimicrobial effects.