In the present study we examined surface and subterranean sites in order to compare the fish fauna composition and characteristics of the physical habitat in a stream located in a Neotropical karst landscape at the São Francisco River basin, Minas Gerais State, Brazil. We investigated the effects of physical habitat characteristics related to river morphology, substrate composition, wood and canopy cover on the fish community and investigated whether subterranean habitats have additional effects responsible for shaping the fish community structure. During the sampling period (dry season; July 2012), 28 species distributed in 3 orders and 9 families were collected (773 individuals in total). Characiformes was the most diverse group in the studied area, with 16 registered species (57% of the sampled fauna). We did not find cave-adapted fish species during the study, although four non-troglomorphic species (two Characiformes and two Siluriformes) were sampled exclusively in the cave. The species composition was possibly affected by environmental differences, showing distinct patterns between the five sites studied. Canopy cover and the percentage of organic matter on the streambed accounted for most of the variation observed in the fish fauna composition (distance-based linear matrix: adjusted R2=0.56; P=0.02). In the system studied, Siluriformes have a greater chance of occupying subterranean habitats, with higher relative abundance and richness inside the cave sites. We believe that number, size and position of subterranean spaces within a karst watershed have additional effects on fish community composition through their selective pressure on species traits.