In signalling interactions, animals can directly address information to a specific individual. Vocal overlapping is such a signalling strategy used in songbirds, anurans, and insects. In songbirds, numerous studies using high rates of song overlap to simulate an escalating situation have shown that song overlapping is perceived as a threatening signal by interacting and by listening (eavesdropping) individuals, indicating a high social relevance of song overlapping. Here we present a playback experiment on nocturnally singing male territorial nightingales (Luscinia megarhynchos). Using three different rates of song overlap (1, 25, or 50%), we tested whether or not lower levels of song overlapping act as a signal of aggressive intent and if birds would increase the intensity of their response with increasing level of song overlapping. Subjects did not vary song duration in response to the different playback treatments but increasingly interrupted their singing with increasing overlap by the three playback treatments. The effects persisted even after the playback ceased to overlap and switched to an alternating singing mode. These results expand on previous studies by showing that song overlapping is interpreted as an aggressive signal even when it is used at low or moderate levels. They suggest that, within the range tested here, increasing levels of song overlapping are perceived to be increasingly aggressive.