It is essential to determine the dominant/indicator species and their ecological preferences to develop a comprehensive bioassessment strategy for rivers. The objective of this work was to provide dependable ecological evaluation techniques for ecosystems that experience significant human-induced disruptions, with the Hari Rud River (a transboundary water resource) serving as a case study during the May (wet) and July (dry) periods of 2023. Canonical correspondence analysis revealed that electrical conductivity (EC), dissolved oxygen, ortho-phosphate (P ), and nitrate (N ) had substantial impacts on the spatial distribution of diatom species in the basin. Relatively pollution-tolerant species, including Nitzschia brevissima, N. capitellata, N. umbonata, N. palea, N. dissipata, and Navicula cryptocephala, had close relationships with EC and P , integrated with Joi Injil and Karbar streams. Of the sampling stations, especially Hari Rud River1 and Hari Rud River2, exhibited pollution-sensitive diatom species, Cymbella excisa, Achnanthidium minutissimum, Diatoma moniliformis, Cymbella affinis, and Meridion circulare. Various eco-regional diatom metrics exhibited distinct scores, indicating a range of ecological status from high to bad in the Lower Hari Rud River basin. European diatom indices revealed good ecological status for Hari Rud River 1 and 4, but poor or bad ecological statuses for Joi Injil and Karbar streams. The findings of the current study emphasize the requirements of autecological studies to understand the regional diatom compositions and their ideal survival ranges in different locations before considering using non-regional diatom indices to evaluate the ecological status of lotic systems.