At present, anaerobic fermentation with waste has become a new utilization direction for the production of WA, and it has advantages for chemical platforms in replacing non-renewable resources that use petrochemicals as raw materials. In this experiment, the pH value, gas production, WA production, bacterial and fungal community structure of rumen fermented wheat straw with ethanol or lactic acid in vitro within 8 transfers (3-day interval) were investigated. Ethanol significantly increased the pH value (P < 0.05), while ethanol and lactic acid significantly decreased the gas production (P < 0.05). Ethanol and lactic acid addition increased the production of valeric acid and caproic acid (P < 0.01). The fermentation of ethanol and lactic acid addition showed marked differences in bacterial composition. Clostridium and Ruminococcus genera were the main bacteria producing caporic acid in ethanol culture, while Megasphaera genus was the main bacteria producing valeric acid in lactic acid culture. The relative abundance of Wallemia genus was positively correlated with valeric acid yield (P < 0.05). This result provides reference data that wheat straw co-cultured with ethanol or lactic acid in vitro increases valeric acid and caproic acid production, and bacteria plays more important role than fungi.