Survival of inoculants in grass silages may enable them to improve the quality of silages through enrichment with polyunsaturated fatty acids (PUFAs). The effects of 2 inoculants, Enterococcus faecium 2/3s (EF2/3s) and E. faecium 26/42 (EF26/42), on nutrient composition, fermentation parameters, and fatty acid content in grass silages during ensiling (111 days) of fresh grass (G) were examined under laboratory conditions. The G [285 g of dry matter (DM) kg-1] was ensiled in 36 polyethylene jars (1 L) divided into 3 × 12 sets per treatment and ensiled at 21 °C for 111 days. The 3 silage treatments used were: 1) grass without inoculant (GS, control), 2) grass inoculated by the strain EF2/3s (GS+EF2/3s), and 3) grass inoculated by the strain EF26/42 (GS+EF26/42). The inoculant strains were sufficiently established during ensiling and reached 4.62 log10 cfu g-1 for EF2/3s and 3.76 log10 cfu g-1 for EF26/42 on day 111 of ensiling. Crude protein contents were G > GS+EF2/3s > GS+EF26/42 > GS (129, 110, 109, and 100 g kg-1 of DM, respectively). The lactate-to-acetate ratios were GS < GS+EF26/42 < GS+EF2/3s (2.82, 4.20, and 4.70, respectively). Concentrations of a-linolenic acid and g-linolenic acid were highest in grass before ensiling (P < 0.001). Higher isomer C18:2 (9,11) content (P < 0.01) was detected in GS+EF2/3s and GS+EF26/42 than in GS. Nutritional manipulation associated with Enterococcus faecium EF2/3s and EF26/42 inoculation of GS resulted in better quality of silages based on lower lactate (GS+EF26/42) and a greater lactic-to-acetic acid ratio (GS+EF2/3s and GS+EF26/42). This might positively affect PUFAs and their isomers.