Evening primrose (Oenothera biennis L., Onagraceae) seeds oil has great economic importance due to its wide industrial application, mainly for medicines and nutraceutics. However, to our knowledge, it remains almost unexplored regarding development of innovative formulations, such as nanoemulsions. On the present study, required Hydroprophile–Lipophile Balance of evening primrose seeds oil was determined (HLB 12) and a stable nanoemulsion (Day 1: mean droplet size: 214.3±0.69nm, polydispersity index: 0.253±0.012. Day 7: mean droplet size: 202.8±0.23nm, polydispersity index: 0.231±0.008) was achieved. Moreover, pseudo-ternary diagram allowed delimitation of nanoemulsion region, contributing to nanobiotechnology of natural products.