The critically endangered Claire's mouse lemur, only found in the evergreen rain forest of the National Park Lokobe (LNP) and a few lowland evergreen rain forest fragments of northern Madagascar, was described recently. The present study provides the first quantified information on vocal acoustics of calls, sound associated behavioral context, acoustic niche, and vocal activity of this species. We recorded vocal and social behavior of six male-female and six male-male dyads in a standardized social-encounter paradigm in June and July 2016 at the LNP, Nosy Bé island. Over six successive nights per dyad, we audio recorded and observed behaviors for 3 hr at the beginning of the activity period. Based on the visual inspection of spectrograms and standardized multiparametric sound analysis, we identified seven different call types. Call types can be discriminated based on a combination of harmonicity, fundamental frequency variation, call duration, and degree of tonality. Acoustic features of tonal call types showed that for communication, mouse lemurs use the cryptic, high frequency/ultrasonic frequency niche. Two call types, the Tsak and the Grunt call, were emitted most frequently. Significant differences in vocal activity of the Tsak call were found between male-female and male-male dyads, linked primarily to agonistic conflicts. Dominant mouse lemurs vocalized more than subdominant ones, suggesting that signaling may present an honest indicator of fitness. A comparison of our findings of the Claire's mouse lemur with published findings of five bioacoustically studied mouse lemur species points to the notion that a complex interplay between ecology, predation pressure, and phylogenetic relatedness may shape the evolution of acoustic divergence between species in this smallest-bodied primate radiation. Thus, comparative bioacoustic studies, using standardized procedures, are promising to unravel the role of vocalization for primate species diversity and evolution and for identifying candidates for vocalization-based non-invasive monitoring for conservation purposes.