Humic substances (HS) are the most abundant forms of natural organic matter on the earth surface. Comprised of decomposed plant and animal materials rich in carbon, oxygen, hydrogen, nitrogen, and sulfur complexes, HS facilitate global carbon and nitrogen cycling and the transport of anthropogenic contaminants. While it is known that HS also interact with organisms at different trophic levels to produce beneficial and harmful effects whether HS exert these biological effects through accumulation remains unknown. Current radiolabeling techniques, which only detect the amount of accumulated radiolabels, cannot visualize the transport and accumulation behavior of HS. Here, using a label-free method based on pump-probe microscopy, we show HS entered the protozoan Tetrahymena thermophila, zebrafish embryos, and human cells and exerted direct effects on these organisms. HS accumulated in the nucleus of T. thermophila, chorion pore canals of zebrafish embryos, and nucleus of intestinal and lung cells in a concentration- and time-dependent way. Epigenetic and transcriptomics assays show HS altered chromatin accessibility and gene transcription in T. thermophila. In zebrafish larvae, HS induced neurotoxicity, altering spontaneous muscle contraction and locomotor activity. Detailed images showing HS accumulation in our study reveal new insights on the ecological and environmental behavior of HS.