In a pot experiment, we explored the regulatory pathways through which melatonin (MT) protects wheat growth and grain yield loss from waterlogging injury. Two wheat cultivars, Yangmai 18 and Yannong 19, were exposed to seven days of soil waterlogging at flowering. Melatonin (100 μmol·L−1) was sprayed before and after waterlogging to explore its regulation on root growth, photosynthetic characteristics, dry matter accumulation, and grain yield. Soil waterlogging intensified malondialdehyde (MDA) and O2− production rates in wheat tissues, impairing leaf photosynthesis, biomass accumulation, and final grain yield formation. In this study, the roots waterlogged at 7 days after anthesis (DAA) accumulated 20.9%, 76.2%, 17.6%, 28.5%, and 5.6% higher MDA content, O2− production rate, pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH) activities, respectively, in Yangmai 18, and 25.7%, 74.8%, 35.8%, 70.8%, and 30.7% higher in Yannong 19, respectively, compared with their respective non-waterlogged controls. Further, Yangmai 18 achieved a maximum net photosynthetic rate (Pn) reduction of 22.1% at 7 DAA, while the maximum Pn reduction of Yannong 19 was 27.4% at 14 DAA, respectively, compared with their respective non-waterlogged plants. Thus, waterlogging decreased total dry matter accumulation, 1000-grain weight (TGW), and total grain yield by 14.0%, 13.8%, and 16.2%, respectively, in Yangmai 18, and 16.0%, 8.1%, and 25.1%, respectively, in Yannong 19. Our study also suggests that exogenously applied melatonin can protect wheat root tissues from waterlogging-induced oxidative injury by upregulating antioxidant enzymes and sustaining leaf photosynthesis. The plants treated with melatonin showed better water status and less oxidative damage, which was conducive to maintaining a higher photosynthetic capacity, thereby improving the waterlogging tolerance of wheat. For example, compared with waterlogged plants, melatonin treatments significantly reduced MDA content, O2− production rate, PDC, LDH, and ADH activities by 7.7%, 25.4%, 2.6%, 32.1%, and 3.2%, respectively, in Yangmai 18, and 6.7%, 17.9%, 4.1%, 22.0%, and 15.3%, respectively, in Yannong 19. MT treatments significantly increased total dry matter accumulation, TGW, and yield by 5.9%, 8.7%, and 14.9%, respectively, in Yangmai 18, and 3.2%, 7.3%, and 26.0%, respectively, in Yannong 19.