Single-pulse femtosecond laser patterning of nitrobiphenylthiol monolayers on Au-coated Si substrates at = 800 nm, < 30 fs and ambient conditions has been investigated. After laser processing wet etching experiments are performed. Laser irradiation reduces the chemical resistance of the coating. In particular, the monolayer acts as a positive-tone resist. Burr-free pattern transfer is feasible at laser pulse fluences between 1 and 2.7 J/cm2. Minimum structure sizes at a 1/e laser spot diameter of about 1 m are close to 300 nm, i.e. sub-wavelength processing is demonstrated. Noteworthy, however, no indications for negative-tone resist properties of processed monolayers are evident, that is, cross-linking of the biphenyl moieties, if at all, is marginal. Also, complementary labeling experiments provide no evidence for chemical transformation of the nitro end groups into amine functionalities. Perspectives of resonant fs-laser processing in exploiting the particular prospects of nitrobiphenylthiol monolayers as negative-tone resists and chemically patternable platforms are discussed.