Sertoli cells are essential for germ cell development and function. Their disruption by endocrine disrupting chemicals (EDCs) or drugs could jeopardize spermatogenesis, contributing to male infertility. Perinatal exposure to EDCs and acetaminophen (APAP) disrupts male reproductive functions in animals and humans. Infants can be exposed simultaneously to the dietary soy phytoestrogen genistein (GEN) and APAP used for fever or pain relief. Our goal was to determine the effects of 10–100 µM APAP and GEN, alone or mixed, on immature Sertoli cells using mouse TM4 Sertoli cell line and postnatal-day 8 rat Sertoli cells, by measuring cell viability, proliferation, prostaglandins, genes and protein expression, and functional pathways. A value of 50 µM APAP decreased the viability, while 100 µM APAP and GEN decreased the proliferation. Sertoli cell and eicosanoid pathway genes were affected by GEN and mixtures, with downregulation of Sox9,Cox1,Cox2, and genes relevant for Sertoli cell function, while genes involved in inflammation were increased. RNA-seq analysis identified p53 and TNF signaling pathways as common targets of GEN and GEN mixture in both cell types. These results suggest that APAP and GEN dysregulate immature Sertoli cell function and may aid in elucidating novel EDC and drug targets contributing to the etiology of male infertility.