The ubiquitous presence of modified mycotoxins such as zearalenone-14-glucoside (ZEN-14G) in agricultural resources presents a critical challenge to livestock safety, specifically regarding their capacity to disrupt lipid metabolism and fermentation in the rumen. This study aimed to evaluate the dose- and time-dependent direct effects of ZEN-14G on rumen fermentation characteristics, nutrient disappearance, and mycotoxin biotransformation under controlled in vitro conditions, and to explore the associated microbial and metabolic responses. An in vitro batch fermentation system was established with three ZEN-14G doses, namely control (CON), low dosage (GL), and high dosage (GH), across three time points (6, 12, and 24 h). Basic fermentation parameters, fat disappearance rates, and ZEN-14G metabolites were quantified, while 16S rRNA gene sequencing and untargeted metabolomics were conducted specifically on 24 h endpoint samples from the CON and GH groups to investigate downstream mechanistic disruptions. The results showed that while baseline pH homeostasis remained unaffected across groups, ZEN-14G dose-dependently inhibited fat disappearance, with the GH group exhibiting a significant reduction throughout fermentation. Targeted quantification confirmed that ZEN-14G was predominantly deglucosylated to free ZEN and reduced to α/β-ZEL. Endpoint multi-omics revealed that although overall α/β-diversity was maintained at 24 h, ZEN-14G induced fine-scale strain-level replacement (12% shared ASVs), marked by the depletion of the key lipolytic bacterium Prevotella sp. R79. Untargeted metabolomics showed marked disruptions in sphingolipid metabolism and linoleic acid oxidation, where the accumulation of cytotoxic oxidized fatty acids (12,13-EpOME) and membrane turnover markers (sphingosine) positively correlated with enriched Bacillota. In summary, under macroscopic acid–base homeostasis, masked ZEN-14G directly impairs ruminal lipid metabolism, alters microbial community structure at the strain level, and perturbs cell membrane lipid turnover strictly within an in vitro system. These findings establish a key mechanistic baseline for masked mycotoxin biotransformation in the rumen, highlighting the necessity for future in vivo feeding trials to fully evaluate their systemic risk profile in ruminants.