The Atlantic Rainforest is among the main biodiversity hotspots in the world, the Yabotí Biosphere Reserve (YBR) being one of the most important remaining areas. Agriculture practices could lead to intensive usage of pesticides resulting in a risk to the environment and human health. Water, suspended particulate matter (SPM), sediment, and fish (Andromakhe paris and Andromakhe saguazu) samples were collected from four streams with different degrees of protection of the YBR in two periods in order to assess the distribution of 18 organochlorine pesticides. Legacy and current-use pesticides were found in the different environmental matrices of the stream headwaters in non-anthropized areas within the buffer zone that drains the intangible area. A similar occurrence pattern of pesticides was found in all matrices. Levels of DDTs (<3.63 ng/L) and endosulfans (<21.8 ng/L) in surface water were above international guidelines for the protection of aquatic life in several streams for both sampling periods. HCHs, DDTs, endosulfans, and chlorpyrifos were detected in SPM and sediments from three streams, while γ-HCH (<60.3 ng/g lipid weight), chlorpyrifos (<698 ng/g lw), p,p´-DDD (<367 ng/g lw), and α-endosulfans (<209 ng/g lw) were detected in fish muscle in several streams. Chlorpyrifos and endosulfans were associated with current use, while DDx/DDT ratios suggested an old use. The concentration of pesticides found would not represent a risk to human health; however, it highlights the need to establish better regulation and action guidelines to reduce the anthropogenic effect on natural reserves.