Saccharomyces cerevisiae is a yeast strain frequently incorporated into ruminant feed formulations to enhance nutritional value. Despite its widespread use, this species demonstrates weak biomass formation when cultivated with carbon substrates and exhibits minimal cellulase enzyme synthesis. The current investigation proposed that native ruminal yeasts may proliferate more efficiently and generate cellulases capable of degrading fibrous material. Therefore, the study aimed to isolate, characterize, and evaluate yeasts obtained from the rumen content of Holstein Friesian steers with respect to their biomass yield and cellulolytic capacity. Fermentation media were optimized using sugarcane molasses as the carbon source and urea as the nitrogen source. Two fistulated crossbred Holstein Friesian steers, each averaging 350 ± 20 kg body weight, served as the donors of ruminal fluid for yeast isolation and screening. The research comprised two experimental phases. The first utilized a 12 × 3 × 3 factorial arrangement within a completely randomized design to analyze biomass accumulation and carboxymethyl cellulase (CMCase) activity. Factor A represented both the isolated yeast strains and S. cerevisiae; Factor B was molasses (M) concentration; Factor C was urea (U) concentration. In the second phase, selected yeast strains with promising performance were identified and assessed using a 7 × 4 factorial randomized design. Here, Factor A denoted incubation time, and Factor B referred to the isolated yeast codes: H-Khon Kaen University (KKU) 20 (P. kudriavzevii-KKU20), I-KKU20 (C. tropicalis-KKU20), and C-KKU20 (Galactomyces sp.-KKU20). Aerobic culturing yielded 11 morphologically distinct colonies. Two colony types were recorded: irregular (A, B, C, E, J) and oval (D, F, G, H, I, K). Strains grown in molasses–urea formulations of 15% + 3% (M15 + U3), 25% + 1% (M25 + U1), 25% + 3% (M25 + U3), and 25% + 5% (M25 + U5) demonstrated significantly greater biomass yields than others (p < 0.01). In total, 11 biomass-forming yeasts were identified in the M25 + U1 treatment. Four isolates capable of producing cellulase enzymes were also detected in M25 + U1 and M25 + U5, while other combinations—M5 + U1, M5 + U3, M5 + U5, M15 + U1, M15 + U3, and M25 + U3—contained 2, 3, 1, 2, 1, and 2 isolates, respectively. The three strains, H-KKU20, I-KKU20, and C-KKU20, were chosen for their high biomass productivity. Identification showed that H-KKU20 and I-KKU20 corresponded to Pichia kudriavzevii-KKU20 and Candida tropicalis-KKU20, respectively, while C-KKU20 belonged to Galactomyces sp.-KKU20. The greatest cell densities were achieved by P. kudriavzevii-KKU20 (9.78 and 10.02 Log cells/mL) and C. tropicalis-KKU20 (9.53 and 9.6 Log cells/mL) after 60 and 72 h of incubation, respectively. S. cerevisiae displayed peak ethanol synthesis at 76.4, 77.8, 78.5, and 78.6 g/L following 36, 48, 60, and 72 h, respectively (p < 0.01). The lowest residual reducing sugar concentrations were observed in P. kudriavzevii-KKU20, with 30.6 and 29.8 g/L recorded at 60 and 72 h. In summary, 11 yeast isolates were recovered from rumen fluid, among which P. kudriavzevii-KKU20, C. tropicalis-KKU20, and Galactomyces sp.-KKU20 emerged as the most promising, with
