This study investigated the effects of direct-fed microbials (DFM) containing Schleiferilactobacillus harbinensis (lactic acid bacteria, LAB) and Pichia kudriavzevii (yeast), applied in different combinations and doses, on rumen fermentation, nutrient digestibility, and microbial populations using an in vitro system. A 3 × 4 factorial design was used, with three LAB:yeast ratios (A1: 1:1, A2: 1:3, A3: 3:1) and four DFM inclusion levels (1%, 2%, 3%, and 4% v/v) with three replicates per treatment. The fermentation substrate consisted of 60% Pennisetum purpureum and 40% concentrate, incubated with goat rumen fluid for 48 h. Data were analysed using generalised linear mixed models, with DFM ratio and dose as fixed effects and their interaction as a random effect. DFM combinations did not alter ruminal pH or overall fermentation; however, the 1:1 ratio (A1) significantly increased butyrate, iso-butyrate, and iso-valerate concentrations (P < 0.05). Increasing DFM dose (up to 4%) elevated NH3 concentration, total short-chain fatty acids, propionate and butyrate proportions, and reduced the acetate-to-propionate ratio (P < 0.05). Nutrient digestibility improved with higher DFM doses, with the 4% level resulting in the highest in vitro dry matter digestibility, in vitro organic matter digestibility, and in vitro crude fibre digestibility values (P < 0.05). Microbial analysis showed that the 1:1 ratio increased Prevotella ruminicola and Butyrivibrio fibrisolvens, whereas higher doses generally stimulated beneficial microbes and reduced methanogen populations (P < 0.05). In conclusion, a 1:1 combination of S. harbinensis and P. kudriavzevii at a 4% inclusion level improved fermentation profiles, nutrient digestibility, and microbial populations in vitro. These findings highlight the importance of both microbial ratio and dose in optimising DFM formulations to support rumen function.