Multiple functions of von Willebrand factor (VWF), its structural complexity and numerous mutations throughout the whole VWF gene impact the clinical heterogeneity of von Willebrand disease (VWD) and make its accurate diagnosis and distinction from mild haemophilia A challenging. The aim of the present study was to introduce a comprehensive laboratory diagnostic approach that included screening and specific coagulation assays, as well as molecular analysis of VWF and coagulation factor VIII (FVIII) genes by means of next-generation sequencing (NGS) and additional targeted screening for deletions and duplications using the multiplex ligation-dependent probe amplification method. Of the 83 study participants, 48 were identified with disease-associated mutations in the VWF gene, of whom 19 were classified as type 1 VWD, six as type 3 VWD, while among the 23 patients with type 2 VWD, 15 were assigned as type 2A, seven as type 2B, and in one case type 2N VWD was confirmed. Heterozygous genotype was present in 38 patients, compound heterozygosity in seven patients while three patients were homozygous for mutations within the VWF gene. In total, 36 distinct disease-associated mutations were found within the VWF gene, of which 13 were novel. Four study participants were identified with mutations within the FVIII gene only, while one female participant had mutations both in VWF and FVIII genes. The two siblings who were homozygous for a mutation in the FVIII gene were diagnosed with mild haemophilia A, while the three heterozygous females were classified as carriers of mild haemophilia A. Two mutations within the FVIII gene were novel. Of the remaining 30 (36 %) study participants without mutations within the VWF and FVIII genes, nine had VWF activity below 50 % and were classified as “low VWF“. The present study revealed considerable genetic heterogeneity among patients with VWD in Croatia. The application of simultaneous sequencing of whole VWF and FVIII genes by means of NGS was proven as a valid approach for differential diagnosis of VWD subtypes, as well as for unambiguous distinction of VWD from mild haemophilia A. This study provided basis for appropriate patient treatment and care, as well as enhanced the understanding of the underlying molecular pathophysiology of the bleeding phenotype in the evaluated patients.