In spite of great developments in the agricultural field and plant productivity in the last decades, the concern about the control of agricultural pests is still continuous. However, pest management is expected to have more effects from nanomaterials by providing innovative solutions. The current study confirms the biotransformation of copper nanoparticles (CuNPs) using a cell-free culture extract of metal copper-resistant bacteria Pseudomonas fluorescens MAL2, which was isolated from heavy metal-contaminated soils collected from Sharqia Governorate, Egypt. The local screened bacterial isolate, Pseudomonas fluorescens MAL2, is similar to Pseudomonas fluorescens DSM 12442T DSM. After optimization of growth conditions, F-Base medium was found to be the best medium and pH 7, temperature 35 ◦C, concentration of CuSO4·5H2O 300 ppm, 10 mL supernatant: 40 mL CuSO4·5H2O (300 ppm), and reaction time 90 min were recorded as the best growth conditions to the fabrication of CuNPs. The formed CuNPs were characterized using initially visual observation of the change in the color of the reaction mixture from blue color to the dark green as an indication of CuNPs biotransformation. Then, UV–Vis spectroscopy showed a maximum absorption at 610 nm under the optimum conditions performed. Transmission Electron Microscopy (TEM) revealed the formation of spherical aspect with size ranges from 10:70 nm; moreover, Energy Dispersive X-ray spectroscopy (EDX) indicated the presence of CuNPs and other elements. In addition, the presence of alcohols, phenols, alkenes, and amines is confirmed by Fourier-Transform Infrared spectroscopy (FTIR) spectroscopy analysis. Dynamic Light Scattering (DLS) supported that the Zeta-average size of nanoparticle was 48.07 with 0.227 PdI value. The Zeta potential showed -26.00mV with a single peak. The biosynthesized CuNPs (Bio CuNPs) showed toxicity against the stored grain pest (Tribolium castaneum), where LC50 value was 37 ppm after 5 days of treatment. However, the negligible effect was observed with chemical synthesis of CuNPs (Ch CuNPs) at the same concentration. The results suggest that Bio CuNPs could be used not only as a biocontrol agent, but also as an ecofriendly and inexpensive approach for controlling the stored grain pests.