The root cause of autoantibody formation against factor VIII (FVIII) in acquired hemophilia A (AHA) remains unclear. We aimed to assess whether AHA is exclusively associated with autoantibodies towards FVIII, or whether patients also produce increased levels of autoantibodies against other targets. A case-control study was performed enrolling patients with AHA and age-matched controls. HEp-2 immunofluorescence was applied to screen for anti-nuclear (ANA) and anti-cytoplasmic autoantibodies. Screening for autoantibodies against extractable nuclear antigens (ENA) was performed by enzyme immunoassay (EIA) detecting SSA/Ro, SSB/La, U1RNP (70 kDa, A, C), Scl-70, JO-1, centromere B, Sm, double-stranded DNA, and alpha-fodrin (AF). AHA patients were more often positive for ANA than control patients (64% vs. 30%, odds ratio [OR] 4.02, 1.98-8.18) and had higher ANA titers detected than controls. Cytoplasmic autoantibodies and anti-AF IgA autoantibodies were also more frequent in AHA patients compared to controls. Autoantibodies against any target other than FVIII were found in 78% of AHA patients compared to 46% of controls (OR 4.16, 1.98-8.39). Results were similar preforming sensitivity analyses (excluding either subjects with autoimmune disorders, cancer, pregnancy, or immunosuppressive medication at baseline) and in multivariable binary logistic regression. To exclude that autoantibody staining was merely a result of cross-reactivity of anti-FVIII autoantibodies, we tested a mix of 7 well-characterized monoclonal anti-FVIII antibodies. These antibodies did not stain HEp-2 cells used for ANA detection. In conclusion, a diverse pattern of autoantibodies is associated with AHA, suggesting that a more general breakdown of immune tolerance might be involved in its pathology.