Evening primrose (Oenothera biennis L.) is a valuable medicinal plant known for its oil, which is rich in gamma-linolenic acid. However, the productivity and quality of evening primrose can be significantly affected by drought stress, a common challenge in arid and semi-arid regions. Water deficit stress not only limits plant growth and development but also affects physiological and biochemical processes. To mitigate the adverse effects of drought, the use of chemical and biological fertilizers has been explored. Mycorrhizal fungi and Azospirillum are known for their ability to enhance plant resilience to abiotic stresses, including drought, by improving nutrient uptake and promoting plant growth. Nitrogen and phosphorus (N+P) are essential nutrients that play crucial roles in plant metabolism and stress tolerance. This study aimed to assess the impact of mycorrhizal fungi, Azospirillum, and N+P on evening primrose under drought conditions. One experiment was conducted in two locations, Tehran (semi-arid) and Varamin (arid) in 2014 and 2015, using a greenhouse for growing plants and through field transplantation. Using a split factorial design within a randomized complete block layout, three irrigation regimes (50 %, 40 %, and 30 % of field capacity) constituted the main plots, while the subplots included three chemical fertilizer treatments (none, 50 %, and 100 % of N+P) and four biological fertilizer treatments (non-inoculated and inoculated with mycorrhizal fungi and Azospirillum) in factorial combinations. The study revealed that drought, both moderate and severe, resulted in reduced plant height, shoot dry weight, leaf area index, seed yield, harvest index, phosphorus content, relative water content, and oil yield. Notably, the highest water use efficiency occurred under moderate water deficit stress. Severe water deficit stress, however, led to the highest root dry weight, root ratio to shoot dry weight, and catalase activity. The application of chemical and biological fertilizers alleviated the negative effects of water deficit stress on plant growth and yield. Evening primrose water use efficiency increased under moderate water deficit stress, particularly when coupled with the application of chemical (N+P) and biological (mycorrhizal fungi and Azospirillum) fertilizers. The study emphasized the positive impact of mycorrhizal and Azospirillum inoculation on enhancing evening primrose growth under water deficit stress.