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The nutrient concentration of fruits and vegetables in the U.S.A. has declined in the past 50–70 years. Crop management practices utilizing on-farm inputs are thought to increase crop nutritional quality, but few studies have evaluated this under long-term side-by-side trials. An experiment was conducted from 2004 to 2005 at Rodale Institute’s long-term Farming Systems Trial to investigate the nutritional quality of vegetables under organic manure (MNR) and conventional (CNV) farming systems, with or without arbuscular mycorrhizal fungi (AMF) treatment. AMF reduced the vitamin C content in carrots in both systems in 2004, but the reduction was 87% in CNV and 28% in MNR. AMF also reduced antioxidants in carrots in both CNV and MNR. This trend was likely due to the suppression of native AMF colonization by the non-native AMF inoculum used. Between 2004 and 2005, MNR increased the vitamin C in green peppers by 50% while CNV decreased the vitamin C in red peppers by 48%. Tomatoes under MNR had a 40% greater vitamin C content compared to CNV in 2005. The vegetable yield declined between 2004 and 2005, except for tomato, where the yield increased by 51% and 44% under CNV and MNR, respectively. In general, MNR tended to increase the nutrient concentration of vegetables compared with CNV, while the AMF effects were inconclusive.