The Fusarium mycotoxins deoxynivalenol (DON) and fumonisins (FUM) are common contaminants of dairy cow rations. Despite the rumen’s ability to degrade these mycotoxins to some extent, especially high producing cows are sensitive to these mycotoxins. The main site of DON and FUM detoxification besides the rumen is the liver. Therefore, the aim of this study was to investigate the effect of a contamination level of DON and FUM below US and European Union guidance levels on the performance and liver enzyme activities of dairy cows. In a randomized block design, 12 Holstein cows in mid-lactation (114 ± 16 d in milk) were fed either a negative control TMR (CTR) or a TMR with a low contamination level of Fusarium mycotoxins (0.4 mg/kg DON, 1.1 mg/kg FUM) (MTX). Each of the 3 experimental periods consisted of a 3-week challenge period followed by a 2-week clearance period. Milk production was recorded daily and blood samples were collected from the jugular vein at the beginning (d 0), on d 14 and d 21 of each experimental period. Data were analyzed as repeated measurements in a completely randomized design using the MIXED procedure of SAS (SAS 9.4 TS, 2018). The mycotoxin treatment had a significant effect (P < 0.05) on the milk production. CTR animals produced on average 37.94 kg/d whereas MTX animals produced on average 36.37 kg/d (−1.57 kg/d). Liver enzyme activities in blood increased in MTX-treated cows after 3 weeks. Aspartate amino transferase (AST) activity was significantly higher (P < 0.05) in MTX treated animals (117.1 U/L) compared with CTR (106.6 U/L). While bilirubin was significantly lower in the MTX-group (1.39 µmol/L compared with 1.61 µmol/L in CTR), both alkaline phosphatase (ALP) and albumin were not affected by the MTX treatment. γ-Glutamyl transferase (GGT) activity tended to be higher (P = 0.059) in MTX-treated animals (30.7 U/L) compared with CTR animals (29.1 U/L). Considering that AST and GGT are commonly used as an indicator for hepatic lesions and function it can be concluded that dietary DON and FUM levels that comply with legal limits showed a negative effect on the milk yield and liver enzyme activities in blood of dairy cows.