Swine slurry is a common agricultural fertilizer in many countries. However, its long-term use in large amounts can cause excess nutrient accumulation, alter soil compounds, and potentially influence critical microbial populations such as arbuscular mycorrhizal fungi, which have important roles in plant nutrition and soil sustainability. This work determined if arbuscular mycorrhizal status, external mycelium, and glomalin-related soil protein content were affected by long-term swine slurry application to different soil tillage systems. The experiment was conducted on a clayey oxisol, in southern Brazil. Swine slurry (0, 30, 60, 90, and 120 m3 ha−1 y−1) was applied for 15 years to conventional tillage and no tillage soil prior to the summer (soybean or maize) and winter (wheat or oats) crop seasons. Swine slurry decreased mycorrhizal root colonization, spore number, and total external mycelium. Swine slurry increased active external mycelium and both easily extractable and total glomalin-related soil protein. No-tillage soil had more glomalin-related soil protein than conventional tillage soil. The most significant response variables were root colonization, easily extractable glomalin-related soil protein, and total external arbuscular mycorrhizal mycelia. Long-term application of swine slurry in this environment, even at high rates, did not adversely affect crop yield but did influence arbuscular mycorrhizae fungi and their products in the soil environment. Benefits of swine slurry application for crop nutrition must be weighed against potential adverse consequences for the size, activity, and benefits of the mycorrhizal community to subsequent annual crops.