The work aims to study the effects of phase structure on nano-mechanical strength of octadecyltrichlorosilane (OTS) molecular films. The OTS molecular film samples were prepared at the temperature of ice-water mixture and room temperature, respectively. Morphology of two samples was characterized with AFM, and height difference of the films were observed by applying various tip-loading forces. With the increase of tip-loading force, relative height difference of OTS molecular film prepared at the temperature of ice-water mixture was constantly maintained at nearly 2.02 nm; for the OTS molecular film prepared at room temperature, the relative height difference did not decrease until the tip-loading force increased to 800 pN, and was maintained at nearly 1.44 nm as the force increased to 4 nN. The orderly and densely arranged OTS molecular film is of higher nano-mechanical strength while disorderly and loosely arranged OTS molecular film is of lower nano-mechanical strength, and may be subject to compressive deformation provided with high tip-loading force.