This in vitro study investigated the enhancement of rumen bacterial adhesion on a substrate to increase the digestibility of rice straw in Hanwoo cattle. The rice straw was pretreated with enzymes, probiotics, or ammonia, and the effects on the enhancement of bacterial adhesion and fiber degradation were analyzed using in vitro rumen fermentation. Enzyme treatment included spraying of cellulase and xylanase at 40 and 120 U per g of rice straw, respectively; Saccharomyces cerevisiae culture of 1.0×107 CFU was sprayed as a probiotic treatment per gram of rice straw; ammonia was sprayed at 0.3% per gram of rice straw. Following enzyme treatment, Fibrobacter succinogenes formed a higher adhesion colony than the control group (7.26±0.03 and 8.43±0.20) after 6h and 12h of in vitro culture (p<0.05), respectively. Attachment of Ruminococcus flavefaciens also increased following enzyme treatment (p<0.05) after 6 and 12 h compared to that of the control (5.18±0.06 and 6.60±0.15); and R. albus attachment showed a significant increase compared to that of the control (5.94±0.15) after 6 h of incubation (p<0.05). Probiotic treatment increased attachment of F. succinogenes in comparison with untreated rice straw after 6 h and 12 h of fermentation (p<0.05); R. flavefaciens attachment showed an increase only after 6 h of culture (p<0.05); R. albus was not affected. Attachment of F. succinogenes, R. flavefaciens, and R. albus increased with ammonia treatment after 6 h and 12 h (p<0.05). Dry matter digestibility was higher after the enzyme treatment (3.45±0.21 and 7.04±0.09) than in the control group(1.85±0.08 and 3.94±0.04) after 6 and 12 h of in vitro culture (p<0.05), respectively. It was also higher than that of untreated rice straw after probiotic and ammonia treatments (p<0.05). There was an increase in the enhancement levels of bacterial adhesion depending on the type of fibrolytic bacteria following enzyme, probiotic, and ammonia treatment. These treatments improved digestibility. This enhancement is considered to be greater following enzyme and ammonia treatments than with probiotics.