Spectral properties of animal acoustic signals may help individuals to assess the characteristics of rivals and to adjust their competitive strategies in territorial disputes. Thus, we hypothesize that the distribution of energy across frequency bands in anuran calls determine behavioral responses in male-male competition. Using playback experiments, we investigated the relevance of the harmonic calls in the acoustic communication of the treefrog Dendropsophus minutus. We exposed territorial males to three synthetic acoustic stimuli composed of aggressive notes: (1) standard call (all harmonics and peak frequency corresponding to the second band); (2) inverted-energy call (all harmonics and peak frequency corresponding to the first band); and (3) concentrated-energy call (all energy contained in the second harmonic). Males responded aggressively to all stimuli, mainly by increasing the rate and duration of their aggressive notes. However, when exposed to stimuli with different harmonic configurations, males changed the harmonic structure of their own calls, emitting more A- and B-notes with peak power in the fundamental frequency, particularly when exposed to the concentrated-energy call. Our results suggest that male frogs may use the harmonic structure of calls to assess opponents and modulate territorial and aggressive behavior, triggering complex acoustic adjustments. This study contributes to our knowledge about the functions of acoustic traits in amphibian social interactions, and particularly of the presence of harmonics that has received less attention compared to other acoustic properties in the study of animal acoustic communication.