[Background] In recent years, the ruminal methane mitigation potential of nitrooxy compounds has attracted a wide attention, however, limited information is available for the effect of these compounds on anaerobic fungi, an important fibrolytic microorganism in the rumen. [Objective] To investigate the effects of nitroglycerin (NG) on the activity and metabolism of anaerobic fungi and methanogens. [Methods] The fungal mono-cultures (Piromyces sp. F1) and co-cultures with methanogens (Piromyces sp. F1+Methanobrevibacter sp.) were used. A series of doses of NG (0.0、6.6、13.2 and 19.8 μmol/L) were added in these cultures and the metabolic profile, fibrolytic activities and microbial abundances were comparably analyzed. [Results] In co-cultures, the dose of 6.6 μmol/L NG completely inhibited the methane production, resulting in the accumulation of hydrogen and formate (P<0.05). And the acetate production and the activities of xylanase and carboxymethyl cellulase decreased (P<0.05), while the changes of the abundances of anaerobic fungi and methanogens were not observed (P>0.05). In fungal mono-cultures, the dose of 6.6 μmol/L NG decreased the production of formate, acetate and ethanol as well as the activities of xylanase and carboxymethyl cellulase (P<0.05), while did not affect the total gas and hydrogen production (P>0.05). In the two culture systems, as the dosage of NG increased, the inhibitory effect on anaerobic fungi and methanogens was enhanced. [Conclusion] NG had inhibitory effect on methanogens, but also had negative effect on anaerobic fungi.