Nitrobenzoxadiazole (NBD)-labeled sterols are commonly used as fluorescent cholesterol analogues in membrane biophysics. However, some experimental reports have questioned their ability to emulate the behavior of cholesterol in phospholipid bilayers. For the purpose of a detailed clarification of this matter, atomistic molecular dynamics simulations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, containing either cholesterol or one of two fluorescent cholesterol analogues, 22-NBD-cholesterol or 25-NBD-cholesterol, were carried out. It is found that these sterol probes tend to adopt conformations in which their tail-labeled fluorophore is oriented toward the lipid/water interface, with a location similar to that observed in molecular dynamics simulations of other NBD probes. This implies that in these molecules the long sterol axis is no longer aligned with the membrane normal, and preferentially adopts orientations approximately parallel to the bilayer plane. In turn, these stretched conformations, together with NBD-POPC atomic interactions, lead to slowed-down lateral diffusion of both fluorescent sterols, compared to cholesterol. From computation of the deuterium order parameter and acyl chain tilts of POPC chains for varying POPC-sterol distance, it is observed that the local ordering effect of sterol is altered in both fluorescent derivatives. In agreement with reported experimental data, both fluorescent sterols are able to increase the order of POPC at 20 mol % concentration (as some molecules adopt an upright conformation, possibly related to formation of transbilayer aggregates), albeit to a smaller extent to that of cholesterol. Altogether, this study indicates that both 22- and 25-NBD-cholesterol are unable to mimic the most important features of cholesterol's behavior in lipid bilayers.