Scotch tape (sco) is a zebrafish cardiac mutant initially proposed to exhibit a reduced amount of cardiac jelly, the extracellular matrix between the myocardial and endocardial layers. We analyzed scote382 mutant hearts in detail using both selective plane illumination microscopy (SPIM) and transmission electron microscopy (TEM), and observed a fascinating endocardial defect. Time-lapse SPIM imaging of wild-type and mutant embryos revealed significant and dynamic gaps between endocardial cells during development. Although these gaps close in wild-type animals, they fail to close in the mutants, ultimately leading to a near complete absence of endocardial cells in the atrial chamber by the heart looping stage. TEM analyses confirm the presence of gaps between endocardial cells in sco mutants, allowing the apparent leakage of cardiac jelly into the lumen. High-resolution mapping places the scote382 mutation within the fbn2b locus, which encodes the extracellular matrix protein Fibrillin 2b (OMIM ID: 121050). Complementation and further phenotypic analyses confirm that sco is allelic to puff daddygw1 (pfdgw1), a null mutant in fbn2b, and that scote382 is a hypomorphic allele of fbn2b. fbn2b belongs to a family of genes responsible for the assembly of microfibrils throughout development, and is essential for microfibril structural integrity. In scote382 mutants, Fbn2b is disabled by a missense mutation in a highly conserved cbEGF domain, which likely interferes with protein folding. Integrating data obtained from microscopy and molecular biology, we posit that this mutation impacts the rigidity of Fbn2b, imparting a structural defect that weakens endocardial adhesion thereby resulting in perforated endocardium.