Habitat selection by aquatic organisms is dependent on the availability of adequate conditions to support life and the benefits that the habitat provides. Contaminated environments tend to be less attractive to organisms because reduced habitat quality leads to increased maintenance costs. Consequently, reduced colonization of such disturbed habitats is an expected response. However, colonization has been understudied as an ecotoxicological test endpoint, despite its proven ability to assess habitat selection by populations across various taxa. The aim of the present study was to investigate whether previous exposure to copper could alter the colonization behavior of the freshwater prawn Macrobrachium rosenbergii along a non-forced exposure gradient of interconnected copper-contaminated habitats (0 to 500 µg/L) due to physiological and behavioral impairments. To assess this, post-larvae of M. rosenbergii were pre-exposed to 0, 50, 250 and 500 µg/L copper for a maximum period of 48 h. The physiological status and motility of the organisms after pre-exposure to copper were evaluated using behavioral endpoints (swimming activity by video tracking) and biochemical biomarkers (biotransformation, oxidative stress and neurotoxicity). The results indicated that pre-exposure to copper (at concentrations of 0, 50 and 500 µg/L) significantly influenced the median colonization concentration (CC50), which decreased from 270 µg/L to 109 µg/L. None of the assessed swimming parameters (speed, motility rate, exploration rate, and total distance) were affected by the pre-exposure to copper (0, 50 and 250 µg/L). Biochemically, cholinesterase levels were only affected in the prawn population exposed to 250 µg/L of copper. The present study provides a better understanding of the relevance of colonization as an ecotoxicological endpoint for assessing the spatial distribution of populations, including both new inhabitants and previously exposed organisms, in recovering habitats.