Excessive release of engineered nanoparticles (NPs) into the environment can affect the biological and chemical properties of soils. This study evaluated the effects of two commercial metallic copper nanoparticles (Cu-NPs 25 nm; CuNPs/25, and Cu-NPs 40–60 nm; CuNPs/40–60) on enzymatic activity and nutrient bioavailability in an agricultural soil. An incubation experiment was conducted using Ultisol soil treated with 500 mg kg⁻¹ CuNPs/25 or CuNPs/40–60, simulating a high-exposure scenario, in the presence and absence of 200 mg kg⁻¹ P (K₂HPO₄). Acid phosphatase, alkaline phosphatase, and β-glucosidase enzymatic activities along with P bioavailability were measured at 0.14, 1, 3, 5, and 8 weeks. The results showed that in most instances, 500 mg kg⁻¹ CuNPs/25 or CuNPs/40–60 significantly decreased the activities of acid phosphatase, alkaline phosphatase, and β-glucosidase. The P bioavailability in soil was lower in the presence of CuNPs/25 than Cu-NPs/40–60. In addition, the Cu²⁺ concentrations in the water-extractable fraction were higher than in the CaCl₂ fraction, and the increase in water-extractable Cu²⁺ concentrations in the soil solution was correlated with a decrease in enzyme activities. Eight weeks of exposure to NPs did not cause any marked changes in organic matter content or electrical conductivity. However, the Cu²⁺-(DTPA) bioavailability in soil treated with 500 mg kg⁻¹ CuNPs/25 and CuNPs/40–60 for 8 weeks increased over 90 times, regardless of the presence or absence of P. However, these findings should be interpreted in the context of a high-exposure scenario for CuNPs and should not be directly extrapolated to environmentally realistic exposure levels.