Linezolid, an oxazolidinone antibiotic, is widely used to treat multidrug-resistant tuberculosis and drug-resistant Gram-positive infections. However, prolonged use is associated with severe hematologic toxicity, the underlying mechanisms of which remain incompletely understood, particularly regarding the role of linezolid metabolites. Our clinical study indicates that elevated exposure to PNU142586, a primary metabolite of linezolid, is associated with an increased risk of linezolid-induced toxicity, even in the absence of renal impairment. To elucidate its mechanism, we identify DNA topoisomerase 2-α (TOP2A) and DNA topoisomerase 2-β (TOP2B) as primary targets of PNU142586 at molecular, cellular, and in vivo levels. PNU142586 disrupts replication and transcription by impeding DNA binding to TOP2A and TOP2B with a favorable conformation for cleavage and by inhibiting adenosine 5'-triphosphate hydrolysis, ultimately leading to antiproliferative and cytotoxic effects, including mitochondrial dysfunction. The present study thus provides mechanistic insight into linezolid-induced hematologic toxicity and offers a foundation for safer antibiotic development and improved clinical monitoring through biomarker identification.