The objective of this research was to conduct in situ measurements of electrical conductivity (EC), pH, dissolved oxygen (DO), and temperature, and collect water samples simultaneously at different depths using an unmanned aerial vehicle (UAV). The UAV system consists of a hexacopter, water sampling cartridges (WSC), and a sensor node. Payload capacity and endurance of the UAV were determined using an indoor test station. The UAV was able to produce 106 N of thrust for 10 min with 6.3 kg of total takeoff weight. The thrust-to-weight ratio of the UAV was 2.5 at 50% throttle. The decision for activating the water sampling cartridges and sensor node was made autonomously from an onboard microcontroller. System functions were verified at 0.5 m and 3.0 m depths in 6 locations over a 1.1 ha agricultural pond. Average measurements of EC, pH, DO, and temperature at 0.5 m depth were 42 µS/cm, 5.6, 8.2 mg/L, and 31 °C, while the measurements at 3 m depth were 80 µS/cm, 5.3, 5.34 mg/L, and 24 °C, respectively. The UAV-assisted autonomous water sampling system (UASS) successfully activated the WSC at each sampling location. The UASS would reduce the duration of water quality assessment and help practitioners and researchers to conduct observations with lower operational costs. The developed system would be useful for sampling and monitoring of water reservoirs, lakes, rivers, and ponds periodically or after natural disasters.