Humic substances (HS) perform a fundamental role in aquatic environments, exhibiting different levels of reactivity in retaining metal ions and organic pollutants. Also, they control the primary production of these ecosystems and act in the carbon sequestering process. In order to improve our understanding vis-à-vis the structural and functional features of HS from aquatic systems, this study aimed to chemically and spectroscopically characterize humic acids (HA) isolated from bottom sediment samples of a stream in a Brazilian subtropical microbasin by elemental analysis, and infrared (FT-IR), ultraviolet and visible (UV-Vis) and solid-state 13C nuclear magnetic resonance (CP-MAS 13C NMR) spectroscopies, thermogravimetry (TG), and scanning electron microscopy (SEM). Although all samples originated from the same environment, the data showed that the HA have distinct chemical and spectroscopic properties, and that the location and characteristics of the sampling points from which the sediments were collected played an important role in the differences observed. Furthermore, vascular plant matter is probably the main contributor to these samples.