We studied the population structure, spatial patterns, and activity patterns of the water opossum (Chironectes minimus) via capture–mark–recapture and radiotelemetry from October 2004 to October 2006 in Atlantic Forest streams in southeastern Brazil. We tested the hypothesis of reproductive seasonality, the usual pattern in neotropical marsupials, by examining recruitment of juveniles. The hypothesis was rejected, supporting the alternative hypothesis that breeding by water opossums was better explained by its habitat characteristics and prey availability than by its phylogenetic ancestry. The observed sex ratio was significantly biased toward males. The home lengths of individual water opossums varied from 844 to 3,724 m. Males had larger home lengths than females, and there were male–male and male–female overlaps. The longer home lengths for males may explain the observed sex ratio bias, because males are more exposed to capture. All individuals used stream stretches with preserved riparian forest, fast-flowing water, and stony substrate. The hypothesis that activity was homogeneously distributed throughout the night was rejected; activity began right after sunset and ended before sunrise, but with higher levels of activity in the first 6 h of the night. Because of anthropogenic alterations of hydrographic basins, water opossums may be threatened by reduction of their main habitat, streams with fast-flowing water and stony substrate.