Three-dimensional (3D) phospholipid monolayers at hydrophobic surfaces are ubiquitous and found in nature as adiposome organelles or in man-made materials such as drug delivery systems. However, the molecular level understanding of such monolayers remains elusive. Here, we investigate the molecular structure of phosphatidylcholine (PC) lipids forming 3D monolayers on the surface of hexadecane nanodroplets. The effects of acyl chain length, saturation, and number of acyl tails per lipid were studied with vibrational sum frequency and second harmonic scattering techniques. We find that 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) lipids form tightly packed monolayers. Upon shortening the tail length to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), more gauche defects are observed. Monolayers of unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and single acyl chained 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) contain more disorder. Despite these variations in the packing, the headgroup orientation remained approximately parallel to the nanodroplet interface. Remarkably, the lyso-PC uniquely forms more diluted and “patchy” 3D monolayers.