Nanofluids have attracted significant attention from researchers due to their ability to significantly enhance heat transfer, especially in jet impingement flows, which can improve their cooling performance. However, there is a lack of research on the use of nanofluids in multiple jet impingements, both in terms of experimental and numerical studies. Therefore, further investigation is necessary to fully understand the potential benefits and limitations of using nanofluids in this type of cooling system. Thus, an experimental and numerical investigation was performed to study the flow structure and heat transfer behavior of multiple jet impingement using MgO-water nanofluids with a 3 × 3 inline jet array at a nozzle-to-plate distance of 3 mm. The jet spacing was set to 3, 4.5, and 6 mm; the Reynolds number varies from 1000 to 10,000; and the particle volume fraction ranges from 0% to 0.15%. A 3D numerical analysis using ANSYS Fluent with SST k-ω turbulent model was presented. The single-phase model is adopted to predict the thermal physical nanofluid. The flow field and temperature distribution were investigated. Experimental results show that a nanofluid can provide a heat transfer enhancement at a small jet-to-jet spacing using a high particle volume fraction under a low Reynolds number; otherwise, an adverse effect on heat transfer may occur. The numerical results show that the single-phase model can predict the heat transfer trend of multiple jet impingement using nanofluids correctly but with significant deviation from experimental results because it cannot capture the effect of nanoparticles.