Bovine mycotic abortion is sporadic and caused by different ubiquitous and opportunistic fungi. Recently, a broad spectrum of bacterial opportunists involved in bovine abortion was revealed by 16S rRNA gene amplicon sequencing. We hypothesized that fungal organisms potentially involved in bovine abortion also might remain undetected by conventional culture. In this retrospective study, we therefore applied fungal internal transcribed spacer 2 (ITS2) region amplicon sequencing to 74 cases of bovine abortion submitted to our diagnostic service. The investigation was complemented by fungal culture and, retrospectively, by data from bacteriological, virological and parasitological analyses and histopathological examination of placentas. Fungal DNA was found in both the placentas and abomasal contents, with 92 fungal genera identified. In 18 cases, >75% of the reads belonged to one specific fungal genus: <i>Candida</i> (n = 7), <i>Malassezia</i> (n = 4), <i>Cryptococcus</i> (n = 3), unidentified <i>Capnodiales</i> (n = 3), <i>Actinomucor</i> (n = 1), <i>Cystofilobasidium</i> (n = 1), <i>Penicillium</i> (n = 1), <i>Verticillum</i> (n = 1) and <i>Zymoseptoria</i> (n = 1) with one case harboring two different genera. By culture, in contrast, fungal agents were detected in only 6 cases. Inflammatory and/or necrotizing lesions were found in 27/40 histologically assessed placentas. However, no lesion-associated fungal structures were detected in HE- and PAS-stained specimens. Complementary data revealed the presence of one or more non-fungal possible abortifacient: <i>Chlamydiales</i> , <i>Coxiella burnetii</i> , <i>Leptospira</i> spp., <i>Campylobacter fetus</i> subsp. <i>fetus</i> , <i>Streptococcus uberis</i> , <i>Escherichia coli</i> , <i>Streptococcus pluranimalium</i> , <i>Bacillus licheniformis</i> , <i>Campylobacter fetus</i> subsp. <i>fetus</i> , <i>Serratia marcescens</i> , <i>Trueperella pyogenes</i> , Schmallenbergvirus, <i>Neospora caninum</i> . The mycobiota revealed by sequencing did not differ between cases with or without a possible infectious etiology. Our study suggests that amplicon sequencing of the ITS2 region from DNA isolated from bovine abortion does not provide additional information or new insight into mycotic abortion and without complementary analyses may easily lead to a false interpretation of the role of fungal organisms in bovine abortion.