Aims Anaerobic rumen fungi (Neocallimastigales) play important roles in the breakdown of complex, cellulose-rich material. Subsequent decomposition products are utilized by other microbes, including methanogens. The aim of this study was to determine the effects of dietary changes on anaerobic rumen fungi diversity. Methods and Results Altered diets through increasing concentrate/forage (50:50 vs 90:10) ratios and/or the addition of 6% soya oil were offered to steers and the Neocallimastigales community was assessed by PCR-based fingerprinting with specific primers within the barcode region. Both a decrease in fibre content and the addition of 6% soya oil affected Neocallimastigales diversity within solid and liquid rumen phases. The addition of 6% soya oil decreased species richness. Assemblages were strongly affected by the addition of 6% soya oil, whereas unexpectedly, the fibre decrease had less effect. Differences in volatile fatty acid contents (acetate, propionate and butyrate) were significantly associated with changes in Neocallimastigales assemblages between the treatments. Conclusions Diet clearly influences Neocallimastigales assemblages. The data are interpreted in terms of interactions with other microbial groups involved in fermentation processes within the rumen. Significance and Impact of the Study Knowledge on the influence of diet on anaerobic fungi is necessary to understand changes in microbial processes occurring within the rumen as this may impact on other rumen processes such as methane production.