Acoustic communication plays a pivotal role in conspecific recognition in numerous animal taxa. Vocalizations must therefore have discrete acoustic signatures to facilitate intra-specific communication and to avoid misidentification. Here we investigate the potential role of echolocation in communication in horseshoe bats. Although it has been demonstrated that echolocation can be used to discriminate among con- and hetero-specifics, the specific acoustic cues used in discrimination are still relatively unknown. Furthermore, the Acoustic Communication Hypothesis proposes that in multispecies assemblages, in which echolocation frequencies are likely to overlap, bats partition acoustic space along several dimensions so that each species occupies a discrete communication domain. Thus, multiple echolocation variables may be used in discrimination. The objective of this study was to investigate the potential of various echolocation variables to function as discriminatory cues in echolocation-based species discrimination. Using habituation-dishabituation playback experiments, we firstly tested the ability of Rhinolophus clivosus to discriminate between echolocation pulses of heterospecifics with either discrete or overlapping frequencies. Secondly, to determine whether R. clivosus could use echolocation variables other than frequency, we investigated its ability to discriminate among echolocation pulses differing in only one manipulated parameter. These test variables were identified by their contribution to the dissimilarity among pulses. Our results suggest that R. clivosus could discriminate readily between species using echolocation pulses with discrete frequencies. When frequencies overlapped, the ability of bats to discriminate was dependant on additional acoustic variables that defined the acoustic space occupied by the test signal. These additional acoustic variables included, but may not be restricted to, sweep rate of the FM and duty cycle. Thus, when echolocation pulses share a similar acoustic domain, bats use several cues to reliably discriminate among heterospecifics.