The photothermal laser patterning of functional organic monolayers, prepared on oxide-free hydrogen-terminated silicon, and subsequent backfilling of the laser-written lines with a second organic monolayer that differs in its terminal functionality, is described. Since the thermal monolayer decomposition process is highly nonlinear in the applied laser power density, subwavelength patterning of the organic monolayers is feasible. After photothermal laser patterning of hexadecenyl monolayers, the lines freed up by the laser are backfilled with functional acid fluoride monolayers. Coupling of cysteamine to the acid fluoride groups and subsequent attachment of Au nanoparticles allows easy characterization of the functional lines by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Depending on the laser power and writing speed, functional lines with widths between 1.1 μm and 250 nm can be created. In addition, trifluoroethyl-terminated (TFE) monolayers are also patterned. Subsequently, the decomposed lines are backfilled with a nonfunctional hexadecenyl monolayer, the TFE stripes are converted into thiol stripes, and then finally covered with Au nanoparticles. By reducing the lateral distance between the laser lines, Au-nanoparticle stripes with widths close to 100 nm are obtained. Finally, in view of the great potential of this type of monolayer in the field of biosensing, the ease of fabricating biofunctional patterns is demonstrated by covalent binding of fluorescently labeled oligo-DNA to acid-fluoride-backfilled laser lines, which--as shown by fluorescence microscopy--is accessible for hybridization.