The interest in the fate of pharmacologically active substances (PASs) in the aquatic environment continually increases. However, little is known about pharmacologically active dyes (PADs) as contaminants of water bodies. PADs are used in medicine, but due to their colouring properties are also applied in the textile, cosmetic and food industries. Their large-scale production and widespread applications have caused these dyes permeate to the aquatic environment. The pharmacological activity and toxicological properties of some of these dyes, caused their occurrence in water should be monitored. Up to now, PADs such as crystal violet, malachite green, methylene blue, rhodamine B, have been determined in the water of Greater China and Iran. However, there is no data on whether PADs pose an environmental problem for water bodies in Poland. Thus, different water samples were collected and analysed by the UPLC-MS/MS method allowing the determination of 20 PADs. The tests showed that dyes such as crystal violet, methyl violet 2 B and rhodamine B were found in 2 out of 36 water reservoirs (0.0122–0.0594 μgL−1). The environmental risk assessment indicated that determined dyes for most model organisms did not pose a risk. Only the presence of methyl violet 2 B (0.0571 μgL−1) was related to a low risk for rohu carp, and crystal violet (0.0122–0.0209 μgL−1) showed a moderate risk for medaka fish. The occurrence of PADs was tested on a larger scale in the water samples collected from different water reservoirs in Poland. Based on obtained results, 96.3% of water samples collected from different water bodies (94.5%) were free from dyes. Thus, it could be stated that generally environmental water of Poland is contaminated with PADs at a low level. On the other hand, the presence of dyes in two samples indicates that PADs permeate the water environment, and their occurrence should be monitored.