This paper describes the formation and structure investigation of Langmuir monolayers and Langmuir-Blodgett multilayers formed from amphiphilic derivatives of 2,5-diphenyl-1,3,4-oxadiazole. The 2,5-diphenyl-1,3,4-oxadiazole group as a functional unit with interesting physical and chemical properties is maintained, while the head group, the length of the alkyl chain and the structure of the coupling unit between aromatic and aliphatic part of these linear short-chain amphiphiles is systematically varied in order to explore the influence of this change on the film forming properties and the stability of Langmuir and Langmuir-Blodgett films. Molecular mechanics simulations are shown by these systematic variations to be suitable for the prediction of optimal chemical structures allowing for a stable stratified molecular packing. The combination of a detailed structure investigation of the multilayers based on scanning force microscopy and X-ray data with molecular mechanics simulations yields an insight into the packing of the molecules and the intermolecular interactions.