(1) Background: Anti-drug antibodies (ADAs) may develop during enzyme replacement therapy (ERT) for Fabry disease and potentially affect biochemical response. Evidence have been derived mainly from males, while comparisons between agalsidase-alpha and agalsidase-beta remain limited by differences in ADA assays. The aim of the study is to evaluate ADA prevalence and titers in patients with Fabry disease treated with agalsidase-alpha or agalsidase-beta and to investigate associations with sex, GLA genotype, residual α-galactosidase A activity, and lyso-Gb3 levels. (2) Methods: This multicenter observational study included 117 patients with Fabry disease receiving ERT (50 males, 67 females), treated with agalsidase-alpha (n = 52) or agalsidase-beta (n = 65). ADAs were measured using an in-house enzyme-linked immunosorbent assay, and lyso-Gb3 by liquid chromatography–tandem mass spectrometry. GLA variants were centrally classified as predicted null-type or predicted non-null-type on the basis of the variant class, and α-galactosidase A activity at diagnosis was normalized to the lower limit of the assay reference interval. Multivariable regression analysis identified factors associated with ADA positivity and lyso-Gb3 levels. (3) Results: ADAs were detected in 55/117 patients. Positivity was more frequent in males than females, with no statistically significant difference between agalsidase-alpha and agalsidase-beta. Predicted null-type variants were associated with higher ADA positivity and titers, particularly in males. In multivariable models, male sex and predicted null-type variants independently predicted ADA positivity, whereas higher normalized α-galactosidase A activity was associated with lower odds. ADA positivity was not independently associated with lyso-Gb3. (4) Conclusions: ADA detection was primarily associated with male sex and predicted null-type GLA variants; no statistically significant association with the agalsidase administered was found. The high prevalence of binding ADAs in females warrants confirmation using orthogonal and functional assays.