Many cellular processes entail the separation of nucleic acid strands. Helicases are involved in the separation of the double-stranded DNA, a process fueled by ATP hydrolysis. We investigated the reaction mechanism of two homologous helicases, the bacterial RecQ and the human RECQ1, in vitro, that is, within confined DNA monolayers. We generate arrays of engineered DNA sequences by atomic force microscopy (AFM) nanografting and monitor the enzyme activity on the surface by means of differential, highly precise AFM measurements of the DNA height variation. The latter is associated with the unwinding action of the enzyme onto the surface-bound DNAs because it arises from the different mechanical properties of single- versus double-stranded DNA that are sensibly detected by AFM. Our results highlight different kinetic behaviors for these enzymes under the same experimental conditions.