For the first time, we have compiled a general list of diatoms for the Zarafshan River consisting of 428 species based on our own research and the literature data. Indicator species for nine water parameters were identified, making up more than 90% of the list. Bioindicators and statistical methods revealed that sections of the river around the city of Samarkand and further in the middle reaches reflect the complexity of the impact of the environment on diatom communities. The surveyed sections of the middle reaches of the river are divided into branches and the dynamics of water parameters and diatom communities are shown from the border with Tajikistan to the confluence of the Zarafshan with the Amu Darya. The indices of organic pollution, S, and toxic impact, WESI, were calculated. They show that there is an increase in salinity and turbidity and a decrease in organic pollution downriver. At the same time, the Navoi section is a source of water acidification. Nutrients and heavy metals, as well as phenol pollution, enter the river from various sources, mainly in the middle reaches of the river. The Zarafshan Nature Reserve in the catchment area of the upper section of the river within Uzbekistan is important for maintaining water quality. Bioindicators show an increase in self-purification, with an increase in the species richness and abundance of diatoms in the middle section of the Zarafshan River. The integrated index of river pollution, RPI, shows that most pollution comes from the northern canal of the river in the middle reaches. A general look at the Zarafshan River catchment basin and the dynamics of the identified water parameters and bioindicator species of diatoms shows that the river ecosystem successfully copes with incoming pollution, including transboundary impacts from Tajikistan. Such a conclusion could not be made based on chemical analysis of the water alone. This allowed us to recommend expanding state monitoring points to the lower section of Karakul while including biological indicators in the observations.