The epidermal growth factor (EGF) receptor (EGFR) is heterogeneously distributed on the cellular surface and enriched in clusters with diameters of tens of nanometers. Multivalent presentation of EGF ligand on nanoparticles (NPs) provides an approach for controlling and amplifying the local activation of EGFR in these clusters. Reactive oxygen species (ROS) have been indicated to play a role in the regulation of EGFR activation as second messengers, but the effect of nanoconjugation on EGF-mediated ROS formation and ROS-induced EGFR activation is not well established. The goal of this manuscript is to characterize the multivalent enhancement of EGF-induced ROS formation and to test its effect on EGFR phosphorylation in breast cancer cell models using gold (Au) NPs with a diameter of 81 ± 1 nm functionalized with two different EGF ligand densities (12 ± 7 EGF/NP (NP-EGF12) and 87 ± 6 EGF/NP (NP-EGF87)). In the EGFR overexpressing cell lines MDA-MB-231 and MDA-MB-468, NP-EGF87 achieved a measurable multivalent enhancement of ROS that peaked at concentrations c ROSmax ≤ 25 pM and that were EGFR and nicotinamide adenine dinucleotide phosphate oxidase (NOX) dependent. NP-EGF12 failed to generate comparable ROS levels as NP-EGF87 in the investigated NP input concentration range (0-100 pM). In cells with nearly identical numbers of bound NP-EGF87 and NP-EGF12, the ROS levels for NP-EGF87 were systematically higher, indicating that the multivalent enhancement is exclusively related not only to avidity but also to a stronger stimulation per NP. Importantly, the increase in EGF-induced ROS formation associated with EGF nanoconjugation at c ROSmax resulted in a measurable gain in EGFR phosphorylation, confirming that ROS generation contributes to the multivalent enhancement of EGFR activation in response to NP-EGF87.