This study analyzes the spatio-temporal variability in the abundance and diversity of epilithic diatoms and their relationship with physical and chemical water variables in a key hydrological system in southern Peru. Samples were collected at 12 sites (lower, middle, and upper reaches of the river) during four campaigns (May, August, November, and December 2019). Physicochemical parameters and community metrics of diatoms were measured, employing multivariate analyses to assess the relationships between environmental and biological variables. Sixty-three species were identified, including Surirella brebissonii, Nitzschia clausii, Ulnaria acus, Achnanthidium minutissimum, and Nitzschia palea. The highest species richness was recorded in the lower reaches. The Shannon index ranged from 1.9 (upper reaches) to 3.59 (middle reaches), while the Simpson index showed greater dominance (0.64) in the upper reaches. Evenness (Pielou index) indicated a homogeneous distribution. Electrical conductivity was significantly correlated with S. brebissonii and N. clausii. Species richness showed spatial differences, and variables such as conductivity and major cations significantly influenced community structure, showing a moderate correlation with the relative abundance of species in the Tambo River basin.