Diffusion of dissolved oxygen through activated sludge flocs was studied, as it represents a potential mechanism for simultaneous nitrification and denitrification in activated sludge systems. Dissolved oxygen profiles through six floc particles collected at different times from a full‐scale activated sludge plant demonstrated that that the dissolved oxygen concentration declines through all floc particles. For larger floc particles (2‐mm diameter and greater), the dissolved oxygen concentration reached near‐zero values at depths depending on process operating conditions. A mathematical model based on diffusion of dissolved oxygen, organic substrate (methanol), ammonia, nitrite, and nitrate through a spherical floc and consumption of dissolved oxygen by heterotrophs and autotrophs accurately predicted the dissolved oxygen profile and required adjustment of only one model parameter—the concentration of heterotrophs. A different dissolved oxygen decline pattern was exhibited for the smaller floc particles characterized, with the dissolved oxygen reaching a non‐zero plateau toward the center of the floc. This pattern was not reproduced with the mathematical model developed and suggests that additional mechanisms are responsible for the transport of dissolved oxygen into the center of these flocs. Implications of these results regarding the occurrence of simultaneous nitrification and denitrification include consideration of the factors that affect floc size and distribution (simultaneous nitrification and denitrification is maximized with larger floc particles), coupling of the International Water Association (London) activated models to predict activated sludge composition with diffusion models to consider intrafloc effects, and the effects of substrate diffusion on the apparent half‐saturation constant for various substrates in activated sludge systems.