The pied butterfly bat, Glauconycteris superba, is endemic to the tropical forest zone of Africa, where it was previously known from only five specimens. Here we report the capture of 10 individuals in two localities of the Democratic Republic of the Congo (Mbiye Island and Yoko forest reserve), and we present the first acoustic data of the species recorded using a conventional microphone and a home-made acoustic system for real time 3D localization. Our morphological comparisons show that females are larger and heavier than males, and that the two sexes exhibit the same fur coloration pattern. We found some individual variations concerning the width of the two lateral white stripes on the belly, and the number and extension of white shoulder-spots. The echolocation recordings show evidence for alternation between two call types (A and B), differing in frequency, bandwidth, and duration. The acoustic signals obtained before captures and after releases revealed important variations depending on the trajectories and environmental conditions. Acoustic characteristics, wing measurements, and the unique black and white fur pattern of G. superba suggest that it is a canopy species able to fly at high speeds. Our findings will be useful for future ecological studies to provide new data on the range, population size, trend and threats of G. superba in order to better assess its conservation status.