Agricultural soils receive a huge number of pollutants that lead to reduce soil quality and increase soil degradation. The aim of this research is to evaluate pollution indices and biochemical properties in soils contaminated with heavy metals in the Middle Nile region, Egypt. The results indicated that the pollution load index (PLI) and potential ecological risk index (PER) ranged from 1.67 to 6.15 and 73.61 to 318.74, respectively. Pollution indices for the studied samples were classified as medium to highly polluted. We note in this study that there is a positive relationship between soil contamination degree (CD) and organic matter (OM) (R2 = 0.938) and total count of microbes (TC) (R2 = 0.940) which indicates the effect of heavy metals on them. No significant differences between all sites in dehydrogenase enzymes activity except for site near the source of pollution (Z1). The increased concentration of heavy metals in the studied sites led to a decrease in the microbial biomass (MBC), bacteria, fungi, and actinomycetes in the soil. This study provides a better understanding of the evaluation of pollution indices in soils contaminated with heavy metals and their impact on biochemical properties and gives the decision-maker the best ways to treat these polluted lands in order to preserve public health.