Ruminants are less susceptible to mycotoxins than monogastrics. However, a long-term intake of a diet contaminated with fumonisins (FUM) can lead to reduced feed intake and a loss in milk production. In this study we investigated the effect of fumonisin B₁ and B₂ (FB₁, FB₂) on rumen fermentation parameters using batch fermentations. Pyrex bottles (n = 15) were inoculated with 100 mL of a mixture containing 50% rumen fluid from bulls, 30% water and 20% synthetic saliva. Culture material of Fusarium verticilloides was added to 7 reactor bottles to achieve an end concentration of 7.2 mg/L FB₁ and 2.8 mg/L FB₂ (FUM treatment). Eight reactor bottles remained untreated (negative control). All reactors were incubated for 24 h at 39°C. Samples of the fermentation broth were taken at 0 h, after 1 h and 24 h of fermentation to determine the concentrations of volatile fatty acids (VFA) and the total bacterial counts by flow cytometry. The pH value was measured directly in the reactors. Statistical analysis was performed using SPSS 19.0. Lactate concentrations were significantly higher in reactors with FUM than in reactors without FUM treatment after 24 h of incubation. In the FUM treated reactors, propionate concentrations were significantly lower after 1 h of incubation and concomitantly, the acetate/propionate ratio was significantly higher. Total bacterial counts were significantly lower in the FUM treated reactors compared with the negative control after 1 h of incubation. The pH value was not significantly different between the treatments. While FB₁ concentrations did not change during the incubation (average concentration = 5.3 mg/L), FB₂ was significantly decreased after 24 h of fermentation (0 h: 1.7 mg/L; 24 h: 1.1 mg/L). In conclusion, the addition of FUM-containing culture material partially affected parameters of rumen fermentation in vitro. The toxicologically most relevant fumonisin FB₁ was not degraded by the rumen microbiota.