Chemical desiccation is widely used in agriculture to anticipate harvest and mitigate the effects of adverse environmental conditions. It is applied to both grains and seeds. Although this practice is widely used, there are still significant gaps in understanding the effects of different herbicide application times on seed quality and plant physiological responses. The objective of this study was to evaluate the effects of different herbicide application times on cowpea, focusing on seed quality, physiological responses, and biochemical composition, including chlorophylls, carotenoids, sugars, and proline, under nocturnal desiccation. In the first experiment, eight herbicides and two mixtures were applied at night: diquat, flumioxazin, diquat + flumioxazin, glufosinate ammonium, saflufenacil, carfentrazone, diquat + carfentrazone, atrazine, and glyphosate. All of the tested herbicides caused a reduction in normal seedling formation, with the diquat + carfentrazone combination resulting in 100% abnormal seedlings. A significant decrease in chlorophyll levels (chlorophyll a: 63.5%, chlorophyll b: 50.2%) was observed using diquat, which indicates damage to photosynthetic processes, while the carotenoid content increased. Total soluble sugars and proline were also negatively impacted, reflecting physiological stress and metabolic changes in seedlings. In the second experiment, three application times were tested with diquat, diquat + flumioxazin, and diquat + carfentrazone. Nocturnal application showed the most significant reduction in chlorophyll levels and increased carotenoid levels. Application at noon and late afternoon also significantly changed the soluble sugar and proline levels. These results indicate that the herbicide application time directly influences the seeds’ physiological quality.