Organophosphate esters (OPEs), used as flame retardants and plasticizers, are present ubiquitously in the environment. Previous studies suggest that exposure to OPEs is detrimental to female fertility in humans. However, no experimental information is available on the effects of OPE mixtures on ovarian granulosa cells, which play essential roles in female reproduction. We used high-content imaging to investigate the effects of environmentally relevant OPE mixtures on KGN human granulosa cell phenotypes. Perturbations to steroidogenesis were assessed using ELISA and qRT-PCR. A high-throughput transcriptomic approach, TempO-Seq, was used to identify transcriptional changes in a targeted panel of genes. Effects on lipid homeostasis were explored using a cholesterol assay and global lipidomic profiling. OPE mixtures altered multiple phenotypic features of KGN cells, with triaryl OPEs in the mixture showing higher potencies than other mixture components. The mixtures increased basal production of steroid hormones; this was mediated by significant changes in the expression of critical transcripts involved in steroidogenesis. Further, the total-OPE mixture disrupted cholesterol homeostasis and the composition of intracellular lipid droplets. Exposure to complex mixtures of OPEs, similar to those found in house dust, may adversely affect female reproductive health by altering a multitude of phenotypic and functional endpoints in granulosa cells. This study provides novel insights into the mechanisms of actions underlying the toxicity induced by OPEs and highlights the need to examine the effects of human relevant chemical mixtures.