The city of Puno in Peru is the largest producer of trout (Oncorhynchus mykiss) using intensive floating cage systems installed in Lake Titicaca. As a result, the increase in diseases and the use of antibiotics to control them during the production cycle has been documented. We study the impact of antibiotics on drinking water, trout tissues and the lake's aquatic ecosystem. Nine antibiotics were monitored: tetracyclines, sulfonamides and fluoroquinolones. The samples were collected randomly and analyzed by liquid chromatography coupled to mass spectrometry and the solid-phase extraction system. The sediment samples and surface water samples contain high concentrations of antibiotics. All sediments contain fluoroquinolones (3.74 mg kg−1) and tetracyclines (3.08 mg kg−1) and the surface water contains fluoroquinolones of up to 408.2 and 652.7 ng L−1 in the dry and rainy seasons respectively (P > 0.05). Drinking water samples from the city of Puno collected at sampling points with Lake Titicaca as a source of drinking water, reached an average of 188.1 and 222.2 ng L−1 of ciprofloxacin in dry and rainy seasons respectively. Complementarily, in trout tissues, it reached 7.8 μg kg−1 in oxytetracycline 8.7 μg kg−1 in sulfatizole, 4.2 μg kg−1 in ciprofloxacin and 3.6 μg kg−1 in sarafloxacin. The presence of these antibiotics in surface water is attributed to the aquaculture activity, in addition to runoff and wastewater, and their presence can have detrimental effects on the aquatic ecosystem, and even affect public health due to the consumption of aquaculture products and drinking water contaminated with antibiotic residues.