There is global demand for novel ecotoxicity testing tools that are based on alternative to animal models, have high throughput potential, and may be applicable to a wide diversity of taxa. Here we scaled up a microplate-based cell-free neurochemical testing platform to screen 800 putative endocrine disrupting chemicals from the U.S. Environmental Protection Agency's ToxCast e1k library against the glutamate (NMDA), muscarinic acetylcholine (mACh), and dopamine (D2) receptors. Each assay was tested in cellular membranes isolated from brain tissues from a representative bird (zebra finch = Taeniopygia castanotis), mammal (mink = Neogale vison), and fish (rainbow trout = Oncorhynchus mykiss). The primary objective of this short communication was to make the results database accessible, while also summarising key attributes of assay performance and presenting some initial observations. In total, 7200 species-chemical-assay combinations were tested, of which 453 combinations were classified as a hit (radioligand binding changed by at least 3 standard deviations). There were some differences across species, and most hits were found for the D2 and NMDA receptors. The most active chemical was C.I. Solvent Yellow 14 followed by Diphenhydramine hydrochloride, Gentian Violet, SR271425, and Zamifenacin. Nine chemicals were tested across multiple plates with a mean relative standard deviation of the specific radioligand binding data being 24.6%. The results demonstrate that cell-free assays may serve as screening tools for large chemical libraries especially for ecological species not easily studied using traditional methods. •Cell-free neurochemical assays can screen large chemical libraries.•We screened 800 chemicals against 3 receptor assays in a fish, mammal, and bird.•453 of 7200 species-chemical-assay combinations were classified as a hit.•Most active chemicals were pharmaceuticals and chemical intermediates.