Acquiring clear and usable audio recordings is critical for acoustic analysis of animal vocalizations. Bioacoustics studies commonly face the problem of overlapping signals, but the issue is often ignored, as there is currently no satisfactory solution. This study presents a bi-directional long short-term memory (BLSTM) network to separate overlapping bat calls and reconstruct waveform audio sounds. The separation quality was evaluated using seven temporal-spectrum parameters. The applicability of this method for bat calls was assessed using six different species. In addition, clustering analysis was conducted with separated echolocation calls from each population. Results showed that all syllables in the overlapping calls were separated with high robustness across species. A comparison between the seven temporal-spectrum parameters showed no significant difference and negligible deviation between the extracted and original calls, indicating high separation quality. Clustering analysis of the separated echolocation calls also produced an accuracy of 93.8%, suggesting the reconstructed waveform sounds could be reliably used. These results suggest the proposed technique is a convenient and automated approach for separating overlapping calls using a BLSTM network. This powerful deep neural network approach has the potential to solve complex problems in bioacoustics.