Tree frogs can attach to smooth and rough substrates using their adhesive toe pads. We present the results of an experimental investigation of tree frog attachment to rough substrates, and of the role of mechanical interlocking between superficial toe pad structures and substrate asperities in the tree frog species Litoria caerulea and Hyla cinerea. Using a rotation platform setup, we quantified the adhesive and frictional attachment performance of whole frogs clinging to smooth, micro-, and macrorough substrates. The transparent substrates enabled quantification of the instantaneous contact area during detachment by using frustrated total internal reflection. A linear mixed-effects model shows that the adhesive performance of the pads does not differ significantly with roughness (for nominal roughness levels of 0-15 µm) in both species. This indicates that mechanical interlocking does not contribute to the attachment of whole animals. Our results show that the adhesion performance of tree frogs is higher than reported previously, emphasising the biomimetic potential of tree frog attachment. Overall, our findings contribute to a better understanding of the complex interplay of attachment mechanisms in the toe pads of tree frogs, which may promote future designs of tree-frog-inspired adhesives.