We provide data on the advertisement call and karyotype of the enigmatic anuran Xenohyla eugenioi from the municipality of Ipirá, State of Bahia, Brazil. The advertisement call is formed by a single multi-pulsed note of 0.111 ± 0.015 s of duration and with dominant frequency of 1033.6–2873.6 Hz along two bands. The species presents 2n = 24 chromosomes and fundamental number = 48. DAPI staining revealed positive labeling on centromeric position in all chromosomes, while FISH using telomeric probe labeled all chromosomal ends. These data shed light on the evolutionary history of the genus and contribute to understanding evolutionary aspects of its sister genus Dendropsophus.