Given the massive and growing environmental impacts of conventional agriculture, humanity needs new methods for growing food that not only meet dietary needs but also provide for multiple ecosystem functions with potential benefits to people and biodiversity. Concepts from ecology and complex adaptive systems suggest that persistent structural heterogeneity and functional diversity are key for supporting biodiversity, ecosystem services, and resilience, but these concepts have not been extensively applied in agriculture, which is still dominated by annual monocropping systems. Perennial agriculture seems to embody these ecological concepts—particularly perennial polycultures that combine a variety of long-lived woody perennial food crops with continuous ground cover. However, our understanding of the benefits and trade-offs of such systems compared to conventional agriculture is limited, especially in temperate climates. Here, we provide a systematic and comprehensive study of the ecological attributes of 14 woody perennial polyculture farm fields to conventional annual and hay fields in the U.S. Midwest, one of the most industrialized food-producing regions in the world. We found that perennial fields had (1) more diverse soil fungal, invertebrate, plant, and bird communities but found no difference in soil bacterial communities; (2) less compacted soil; (3) denser ground cover; (4) more active carbon, organic carbon, and nitrogen and the same available phosphorus in the top layer of soil; and (5) more species of predatory, detritivorous, and herbivorous insects, and approximately fourfold higher abundance of herbivorous insects. Food production from the oldest half of perennial fields was only 14.7% of a regional corn/soy rotation by weight, but increased with the age of the perennial field and had high nutritional diversity. Together, these findings indicate that woody perennial polyculture fields in the U.S. Midwest are characterized by higher biodiversity and ecosystem functions than adjacent conventional fields. Woody perennial polycultures in the temperate north might play a role in the transition of agricultural landscapes toward sustaining both people and nature over the long term.