Freshwater ecosystems are endangered, underfunded and understudied, making new methods such as passive acoustic monitoring (PAM) essential for improving the efficiency and effectiveness of data collection. However, many challenges are still to be addressed with PAM: difficulty in accessing research sites, the logistics of implementing large-scale studies and the invasiveness of data collection. When combined with PAM and other sensing strategies, mobile robotics are a promising solution to directly address these challenges. In this paper, we integrate water surface and underwater acoustic monitoring equipment onto a prototype unmanned aerial-aquatic vehicle (UAAV) capable of sailing and flight (SailMAV). Twelve autonomous sailing missions were run on Lake Vrana, Croatia, during which acoustic data were collected, and the ability of the UAAV to facilitate the collection of acoustic data demonstrated. Data were simultaneously collected using standard recording methods on buoys and banksides to provide a comparative approach. Acoustic indices were used to analyse the soundscape of underwater acoustic data and BirdNET (a deep artificial neural network) was used on water surface datasets to determine bird species composition. Results show higher species richness and call abundance from UAAV surveys and high site dissimilarity owing to turnover between stationary and UAAV methods. This highlights the success of the UAAV in detecting biodiversity and the complementarity of these methods in providing a broad picture of the biodiversity of freshwater ecosystems. Increased bird diversity and underwater acoustic activity in protected areas demonstrate the benefits of protecting freshwater ecosystems; however, site dissimilarity driven by turnover highlights the importance of protecting the entire ecosystem. We show how, by integrating PAM and a UAAV, we can overcome some of the current challenges in freshwater biodiversity monitoring, improving accessibility, increasing spatial scale and coverage, and reducing invasiveness.|In this article, we integrate water surface and underwater acoustic monitoring equipment onto a prototype unmanned aerial-aquatic vehicle (UAAV) capable of sailing and flight (SailMAV), to demonstrate how robotics can facilitate acoustic sensing in freshwater ecosystems. Results show higher species richness and call abundance from UAAV surveys and high site dissimilarity owing to turnover between stationary and UAAV methods. This highlights the success of the UAAV in detecting biodiversity and the complementarity of these methods in providing a broad picture of the biodiversity of freshwater ecosystems. Increased bird diversity and underwater acoustic activity in protected areas demonstrate the benefits of protecting freshwater ecosystems; however, site dissimilarity driven by turnover highlights the importance of protecting the entire ecosystem. We show how, by integrating passive acoustic monitoring (PAM) and a UAAV, we can overcome some of the current challenges in freshwater biodiversity monitoring, improving accessibility, increasing spatial scale and coverage, and reducing invasiveness.image