During silage sampling and aeration, the activity of aerobic microorganisms leads to silage deterioration which is indicated by the degradation of lactic acid, nutrient loss and an increase in silage temperature. This study aimed to investigate microbiological characteristics as well as changes of content in lactic acid and VFA in alfalfa silage ensiled without and with three different concentrations of lactic acid bacteria (LAB) inoculant during a seven-day silage aeration. Alfalfa silage was ensiled with three combined concentrations of homofermentative and heterofermentative LAB inoculant (T2: standard, T3: 1.5x standard and T4: 2x standard) and control without LAB inoculant (T1) in vacuum bags (SmartVac, Status d.o.o.). All of the treatments were sampled in five repetitions. After 124 days of ensilling, the silages were subjected to aeration for 7 days. The change in silage temperatures were measured every 15 minutes during the whole aeration period and were sampled 0th, 3rd and 7th day to determine the number of LAB, yeasts, moulds, and spores of butyric acid (BAB), as well as fermentation parameters. During the entire period of aeration, all the tested silages remained aerobically stable. However, T1 and T4 had 3x higher lactic acid values when compared to T2 and T3, thus showing an unexpectedly pronounced homofermentative activity in T4. Inoculant application had a positive influence on acetic acid content at the end of aeration when compared to the control group (38 – 47 g/kg DM in T2 – T4 vs. 30 g/kg DM in T1) and propionic acid (0.83 – 3.32 g/kg DM in T2 – T4 vs. 0.76 g/kg DM in T1). The highest butyric content was in T3, which also had the highest number of BAB. Inoculant application showed a positive effect on LAB numbers at the end of aeration (about 1.16x more LAB in T2 – T4 ) while yeasts and moulds were below average in all tested silages (yeasts < 3.1 log10 CFU/g, moulds < 4.1 log10 CFU/g). The results show when using an inoculant, it is preferred to use the concentration recommended by the producer, because that concentration enables the optimal activity of homofermentative and heterofermentative LAB and the production of silage with an optimal content of lactic acid and VFA for aerobic stability.