Animals can encode multiple messages using acoustic communication in diverse behavioral contexts. Although the anuran advertisement call might convey different messages to males and females, the role of the calling microhabitat type in intra- and intersexual behavior is unclear. We report advertisement call differences in six of the seven acoustic parameters analyzed for the Brazilian hylid frog Hypsiboas atlanticus, depending on the calling microhabitat type (water or perch). These call differences were aurally distinctive and enabled us to count males calling during the reproductive period, estimating abundance of calling males per microhabitat throughout a given night. We used playback tests to determine whether male behaviors vary as a function of the microhabitat type and whether males stimulated by calls from other males in different microhabitats alter their behavior and acoustic parameters. Advertisement calls were influenced by male body size and temperature. The temperature increased or decreased the call’s parameters according to microhabitat type. Playback stimulus promoted acoustic responses depending on the calling microhabitat, playback type, or both. Males calling from perches also emitted territorial calls and visual displays. There was an inverse relationship between the abundance of perched males and those in the water throughout the night. Our results indicate that the calling microhabitat has an impact on the advertisement call function in H. atlanticus: an advertisement call sent from a perch shows a predominant role in territory establishment, whereas an advertisement call sent from males in water might have a predominant role in attracting mates for reproduction.