Objective: This study was conducted to confirm the effects of new inoculants producing-antifungal or esterase substances on rye silage and its rumen fermentation indices by comparing wild with mutated types.Methods: Rye harvested at dough stage was ensiled into 3 L mini bucket silo (1 kg) for 90 d in triplicate following: distilled water at 20 μL/g (CON); <i>Lactobacillus brevis</i> 100D8 (AT) and its inactivation of antifungal genes (AT-m) at 1.2×10⁵ cfu/g, respectively; and <i>Leuconostoc holzapfelii</i> 5H4 (FD) and its inactivation of esterase genes (FD-est) at 1.0×10⁵ cfu/g, respectively. After silo opened, silage was sub-sampled for the analysis of ensiling quality and its rumen fermentation indices.Results: Among the wild type inoculants (CON vs AT vs FD), FD inoculant had higher (p<0.05) <i>in vitro</i> digestibilities of dry matter and neutral detergent fiber, the total degradable fraction, and total volatile fatty acid in rumen, while AT inoculant had higher (p<0.05) lactate, acetate, and lactic acid bacteria in silage. Silage pH and the potentially degradable fraction in rumen increased (p<0.05) by inactivation of antifungal activity (AT vs AT-m), but lactate, acetate, and lactic acid bacteria of silage decreased (p<0.05). In silage, acetate increased (p<0.05) by inactivation of esterase activity (FD vs FD-est) with decreases (p<0.05) of pH, ammonia-N, lactate, and yeast. Moreover, inactivation of esterase activity clearly decreased (p<0.05) <i>in vitro</i> digestibilities of dry matter and neutral detergent fiber, the total degradable fraction, and total volatile fatty acid in the rumen.Conclusion: This study concluded that FD inoculant confirmed esterase activity on rye silage harvested at dough stage, while AT inoculant could not be confirmed with antifungal activity due to the absence of mold in all silages.