The effect of Rhizobium inoculation and the application of two herbicides (Pendimethalin and Glyphosate) on the growth, physiological, and biochemical parameters of Vigna unguiculata was evaluated in this study. Rhizobium inoculation significantly improved seed germination, shoot, root development, and nodule formation, suggesting its symbiotic role in enhancing plant resilience and productivity. Application of herbicides induced a dose-dependent response with low rates of Pendimethalin and Glyphosate, causing minimal damage to growth and nodulation in combination with Rhizobium. However, root length, nodule number, and nitrogen fixation were adversely affected at elevated concentrations of Glyphosate. Importantly, Rhizobium increased the protein content and nitrogen assimilation, but the herbicide treatments negatively affected these parameters at elevated concentrations. Higher concentrations of Glyphosate also produced higher proline levels, a widely used plant stress indicator, and were detrimental in all cases. The activity of some important enzymes, such as nitrate reductase and hydrogenase, was reduced by increasing doses of the herbicide; however, Rhizobium inoculation significantly reversed the noted impacts. In conclusion, the findings highlight the significance of Rhizobium inoculation in improving the physiological performance and stress endurance of cowpeas under herbicide-induced circumstances and provide further evidence for the rational use of Rhizobium for the sustainable cultivation of legumes in herbicide-treated environments.