Introduction <p>The synergistic function between ferroptosis and tumor-associated macrophages (TAMs) has gained significant attention, but their relationship remains unclear. This study explores their roles and mechanisms in ovarian cancer progression, offering insights for potential therapeutic targets.</p> Methods <p>From public databases, 4410 immune-related ovarian genes and 483 ferroptosis-related genes were collected. Using bioinformatics analysis, CYBB was identified as a key gene strongly associated with macrophage infiltration and ferroptosis. The immune infiltration of CYBB in ovarian cancer was evaluated using TIMER2, TISIDB, linkedOmics, GEPIA2, and TISCH databases. The expression and function of CYBB in ovarian cancer were further validated through in vitro cellular assays. Through establishing an in vitro TAMs co-culture model, we investigated the role of CYBB in modulating macrophage polarization.</p> Results <p>Our findings revealed that CYBB is highly expressed in ovarian cancer and associated with poor prognosis. Single-cell sequencing indicated that CYBB is predominantly expressed in macrophages, and enrichment analysis demonstrated that genes co-expressed with CYBB are primarily involved in macrophage activation. Through in vitro experiments, we found that CYBB influences ovarian cancer cell proliferation and erastin-induced ferroptosis. In TAMs, CYBB knockdown increased ferroptosis-related proteins (FTH-1,FSP1). Notably, CYBB knockout suppressed the expression of M1 macrophage markers while promoting the upregulation of M2 marker expression.</p> Conclusion <p>CYBB has prognostic and clinical significance in Ovarian cancer, playing a key role in the immune microenvironment by regulating macrophage infiltration and ferroptosis.</p>