Baling of alfalfa hay at higher than optimum moisture level can lead to spoilage and a reduction in the nutritive quality of the forage. Organic acids such as propionic acid (PA) have been added to high moisture hay (HMH) to prevent the growth of spoilage microorganisms, but the high rates of application, costs and environmental concerns associated with these preservatives has discouraged hay producers from using them. The objective of this study was to assess the effects of applying Pediococcus pentosaceus (PED) and Pichia anomala (PIC) alone or in combinations with chitinase (CH) at baling on the nutritional quality of high-moisture alfalfa hay using in vitro and in situ procedures. Alfalfa hay was harvested at the mid-bloom stage, wilted to 73–77% DM and baled without (HMH) or with PED, PIC, PED+PIC, PED+CH, PIC+CH, and PA. The control (HMH), PED+CH, PIC+CH and PA and alfalfa hay baled at >85% DM (NMH) were repeated in 3 consecutive yrs. Other treatments were assessed only in single yr. Samples collected 90 d after baling over the 3 yr were assessed in vitro and only 1 yr samples were assessed for rumen degradability. For all 3 yr, in vitro DM disappearance (IVDMD), asymptotic gas production and rate of gas production of HMH, irrespective of treatment, was lower (P < 0.05) than NMH. Among treatments, PED+CH and PIC+CH had higher (P = 0.01) IVDMD than HMH and PA, but only PED+CH had higher (P < 0.01) asymptotic and rate of gas production as compared with HMH and PA in yr 1. The potential degradable fraction of DM did not differ among treatments. However, PED+CH and PIC+CH had a higher (P < 0.01) rapidly degradable fraction than PA. The 24-h in situ NDF digestibility of PED+CH and PIC+CH was higher (P < 0.001) than that of all other treatments including NHM. These results suggest that application of P. pentosaceus or P. anomala combined with chitinase at baling of high moisture alfalfa hay has potential to reduce nutrient losses and to increase fiber digestibility.