The diverse anthropogenic load on the transboundary Irtysh River necessitates an assessment of its ecological state, which was the goal of this work. We conducted this research in July 2023 in the upper and lower reaches of the Kazakh part of the Irtysh basin. We determined transparency; temperature; pH; salinity (TDS); oxygen, N-NO3, N-NO2, N-NH4, PO4, Mn, Fe, Si, Cd, Cu, Zn, Pb, Cr, Co, and Hg contents; permanganate index; and zooplankton variables at 27 stations. We assessed the ecological state of the river by comparing the contents of pollutants with their maximum permissible concentrations (MPCfw), Classification Scales, and bioindications. An excess of MPCfw was detected for N-NO2, Cu, and Fe and locally for Cr and Zn. According to the Classification Scales, most analysed variables corresponded to slightly polluted waters; N-NO2, Cr, and Zn corresponded to moderately and heavily polluted waters. Zooplankton was represented by 82 species, with an average abundance of 6728 individuals/m3, biomass of 2.81 mg/m3, Shannon index of 1.99–2.08 bit, ∆-Shannon of 0.09, and average individual mass of 0.0019 mg. The spatial distribution of abiotic and biotic variables indicated increased organic and toxic pollution downstream in the Irtysh. Potential sources of pollution of the Irtysh basin are discussed.