Scutellaria baicalensis Georgi is a medicinal plant and perennial herb that could be used for treatment of hepatitis, cirrhosis of the liver, cancer, jaundice, anxiety, and nerve disorders. In recent years, to meet the high demand for medicine, the area of Scutellaria cultivation has increased in Hebei province, but the quality has reduced because of non-standard planting practices and improper use of fertilizer and water. Therefore, increased research attention has been paid to improving the yield and quality of S. baicalensis in China and abroad.Arbuscular mycorrhizal (AM) fungi are beneficial soil microbes that can form good symbiotic relationships with over 80% of all vascular plants. Some studies have shown that AM symbiosis is significantly influenced by variations in water and fertilizer conditions. This study focused on the effects of an AM fungus species (Acaulospora bireticulata) on the growth, nutrients, and secondary metabolites contents of S. baicalensis under different water and fertilizer conditions using a pot experiment in a greenhouse. The experimental design included 18 treatments representing a combination of 2 mycorrhizal states, 3 water regimes, and 3 fertilizer application levels. The results showed that AM fungal inoculation significantly promoted growth of the host plant and the infection rate, but these inoculation effects were influenced by the soil water and fertilizer levels. Under different water and fertilizer conditions, AM fungal inoculation improved the protective enzyme activity and content of osmotic adjustment substances, and decreased proline and MDA contents. AM fungal inoculation significantly increased the contents of baicalin and N, P, K, Ca, Mg, Fe, and Zn, and decreased the contents of Mn and Cu. The contents of N and P increased with improved fertilizer levels, and other mineral elements were the highest when the fertilizer level was 0.383 g N, 0.564 g P, and 0.251 g P. Therefore, AM fungi could form a good symbiotic relationship with S. baicalensis. Inoculation of AM fungi on S. baicalensis showed that the best effect under conditions of a relative water content of 50% and a fertilizer level of 0.383 g N, 0.564 g P, and 0.251 g K.