A field experiment was done to investigate the effect of biochar application (0, 1 and 2 t/fed) and arbuscular mycorrhizal fungi (AMF) inoculation (un-inoculation, seed-inoculation and after-germination inoculation) on growth, yield and quality of evening primrose plants (Oenothera biennis L.) during the two seasons of 2021/2022 and 2022/2023 at a private farm in El-Sadat City, El-Menoufia Governorate, Egypt. The results showed that growth parameters, flowering characteristics, carbohydrates content, NPK content, seed yield and fixed oil yield were increased with biochar addition especially at 2 t/fed. The greatest outcomes of AMF inoculation were observed when it was carried out after-germination. Application of biochar by 2 t/fed combined with add AMF after-germination inoculation gave the highest values of growth parameters, flowering characteristics, total carbohydrates content, NPK content, seed yield and fixed oil yield of evening primrose plants. The study concludes that integrating biochar and AMF can significantly enhance the growth (i.e., plant height, number of branches, number of leaves per plant, leaf fresh and dry weights per plant and leaf area), yield (i.e. seed weight per plant and per feddan, fixed oil yield per plant and feddan), and quality of evening primrose, offering a sustainable approach to improving medicinal and aromatic plant production.