Arbuscular mycorrhizal fungi (AMF) form symbiosis with plant roots, improving its establishment, nutrition, and tolerance to adverse soil conditions. In annual crop rotations, some aspects such as the type of N fertilizer and tillage system used can affect the AMF propagule density and its functionality in the following crop. To analyze the effect of the agronomic practices previously mentioned on the persistence and density of AMF propagules, a study in a succession of cereals was carried out. For this, soil previously cropped with wheat (Triticum aestivum L.) and fertilized with urea (NH4+) or sodium nitrate (NO3-) was disturbed in each case by simulating conventional tillage (CT) or maintained without disturbing, simulating no-tillage (NT). It was then cultivated with oat (Avena sativa L.) using the same N sources. Higher densities of AMF active mycelium and colonized root length in the NT soil were observed (76 and 497% higher than in CT soil, respectively, P < 0.001). Moreover, NT + NO3- interaction produced a higher density of AMF spores (75% higher than in the rest of the treatments, P < 0.05), although showing a decrease in comparison with the previous wheat crop. These results suggest that N source and tillage system interact to influence persistence and density of AMF propagules, an important aspect to consider in acidic soils intended for annual crops.