Several previous studies have investigated the skin aging prevention effects of ceramide, hyaluronic acid, and natural or fermented plant materials. Recently, oral administration and dermal application of probiotics or probiotic lysates have shown antiaging effects. The purpose of this study is to optimize the preparation of probiotic lysates with a high concentration of nucleotides and to confirm the effects of probiotic lysates on the skin. Probiotic lysates were prepared by heating at 121°C for various periods with adding of sodium hyaluronic acid. Probiotic lysates of Bifidobacterium longum HDB7072, Lactobacillus paracasei HDB1196, and Lactobacillus acidophilus HDB1014 were applied to normal human epidermal keratinocytes (NHEKs), fibroblast cells, and B16F1 cells, respectively. Cell viability, antioxidant effects, and mRNA expression were evaluated by using MTT assays, DPPH assays, and qRT-PCR. Probiotic lysates prepared by heating the culture medium at 121°C for 2 h with 0.5% sodium hyaluronic acid showed the highest nucleotide concentration. In the three tested skin cells, the cell viability of filtered lysates was similar or higher to that of unfiltered lysates. HDB7072 lysates increased filaggrin expression in NHEKs. HDB1196 lysates showed DPPH radical-scavenging and antiwrinkle effects through the downregulation of matrix metalloproteinase-1 and upregulation of collagen type 1 in fibroblasts. HDB1014 lysates had antioxidant and antimelanogenic effects in B16F1 cells. Cell wall-removed probiotic lysates could be used as novel ingredients to improve skin aging and skin barrier issues.