Habitat segregation is a common phenomenon among anguillids in rivers where multiple species coexist. However, despite the growing need to conserve their river habitats, the mechanisms underlying such segregation remain unknown. Here, we conducted tank experiments to investigate the competitive superiority for habitats between the temperate Japanese eel Anguilla japonica and the tropical Indo-Pacific eel Anguilla marmorata, which co-occur in East Asian subtropical rivers. Using eight interspecific pairs of these two species of comparable total length, we compared the use of a single pipe shelter, recorded over a 24-h experimental period, between solitary and paired conditions. The changes in shelter use patterns between conditions indicated that in the paired condition, the shelter was occupied by A. marmorata in five pairs (62.5%) by the end of the experiment while A. japonica appeared to occupy the shelter in only one pair (12.5%). Furthermore, agonistic behaviors, which occurred mainly at night, such as biting and head-butting, were observed much more frequently for A. marmorata toward A. japonica (mean ± standard deviation: 23.2 ± 36.6 times per individual) than for A. japonica toward A. marmorata (0.4 ± 0.5 times per individual), indicating an asymmetry in aggressiveness. These results suggest that A. marmorata can negatively affect the diurnal habitat use and nocturnal activities of sympatric A. japonica. These findings, combined with those from previous field studies, suggest that A. marmorata may competitively exclude A. japonica from stream habitats with large substrate sizes. This asymmetric competition can contribute to shaping their riverine distribution and composition within rivers where they co-occur.