This study investigated the possible relationship between oxidative stress biomarkers and the levels of d- and l-lactic acid enantiomers, as markers of ruminal activity, during the transition period of dairy cattle. Twenty-two late gestational animals were sampled biweekly around the time of calving, and the data obtained was divided ex post into two stages: (1) prepartum (1 month until calving) and (2) postpartum (to 1 month after calving). Forty animals between the fourth to fifth months of gestation were used as controls. Reactive oxygen species, serum antioxidant capacity, and d- and l-lactates were measured in serum samples. Discrepancies among correlations of both lactic acid enantiomers and oxidative stress biomarkers were found, which may be attributable to the different metabolic pathways of the formers within the organism. In light of our results, ruminal activity is related to the oxidative status of transitional dairy cattle, although with different roles for each enantiomer, i.e., pro-oxidant for d-lactate and antioxidant for l-lactate. These findings are the first step in studying the effect of different nutrition strategies that could modulate the fermentation processes that occur within the rumen. This could improve the oxidative status of the animal and therefore minimize the harmful effects of excessive reactive oxygen species production.