Tetracycline is one of the most worldwide used antimicrobial agents. Unfortunately, it is partially metabolized by human body and a significant amount is excreted unchanged into wastewater. In addition, its resistance to biodegradation during wastewater treatment leads to a persistent occurrence in aquatic environments. There, tetracycline exerts a selective pressure on microbial communities, promoting the emergence and spread of antibiotic resistance genes such as tetA and tetM. Tetracycline concentrations in surface waters could reach up to 20 µg/L. This concentration level has been sufficient to induce sublethal effects in sensitive bacteria and to promote adaptive mechanisms within aquatic microbial populations. The aim of this study was to explore microbial adaptation mechanisms, especially of Vibrio fischeri incubated in presence of 0.3÷5 mg/L tetracycline. Tested tetracycline concentrations were higher than those typically found in the environment, but they were selected to define adaptation limits and clearly observe toxic effects under controlled laboratory conditions such as Vibrio fischeri bioluminescence and optical density (OD₆₀₀). Results revealed significant reductions in both bioluminescence and viability at concentrations as low as 0.6 mg/L and a complete inhibition at 5 mg/L. The Relative Light Units (RLU) / OD₆₀₀, ratio indicated changes in bacterial metabolic efficiency across the tested range. The dose–response patterns were aligned with ecotoxicity thresholds and supported the integration of V. fischeri-based assays into environmental risk assessment frameworks. Chronic exposure results further suggested that sublethal antibiotic levels may influence microbial community structure and resistance development in aquatic ecosystems.