Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.