This study utilized liquid chromatography (LC) alongside Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to explore the dyes and chemical contaminants in Loji River, Indonesia. We tentatively identified a total of 655 contaminants at various confidence level, subsequently classifying them into 22 distinct categories. Of the 54 dyes we detected, 12 corresponded with entries in our specialized in-house database. These 12 dyes were further confirmed by reference standards, matching both retention time (RT) and MS/MS spectra. LC-FT-ICR MS data showed that dyes from printing batik and textile industries are key contributors to river pollution. Particularly noteworthy were two sample locations that displayed substantial contamination, predominantly from azoic and reactive dyes. Additionally, pharmaceuticals were identified as one of the most frequently occurring contaminants, underscoring the inadequacies in the area's sewage management. To corroborate these findings, we conducted physicochemical, phytotoxicity, and acute toxicity tests, all of which verified the harmful effects of the Loji River's water on both the local flora and human populations. Notably, water samples that tested positive for dye contamination exhibited elevated toxicity levels. To the best of our knowledge, this study is pioneering in its molecular-level investigation of dye contamination in Southeast Asian rivers. Our results accentuate the pressing need for both targeted and non-targeted screening methods to identify contaminants in the surface waters of developing nations.