As ruminants in Egypt are mainly fed on lignocellulosic agricultural by-products, so the present study aimed to investigate the fungal cellulolytic activity in absence of cither rumen bacterial or protozoal populations after 12 and 24 hours of in vitro incubation. The rumen contents of five steers were collected immediately after slaughtering, and used to generate three microbial systems, a control system (whole ruminal fluid without chemical treatment to measure activity of all microbial groups), a protozoal+fungal system (protozoal and fungal groups plus antibacterial agent) and a bacterial+fungal system (bacterial and fungal groups plus antiprotozoal agent). The fermentation patterns of cellulose due to the various treatments resulted in three distinct groups of data. Absence of either bacterial or protozoa species had a positive effect on fungal zoospores count, cellulose degradation %, total volatile fatty acids (VFAs) concentrations but negatively affected methane production without any alteration in cither ammonia nitrogen concentration or CO2 production. Nevertheless, the positive effects achieved by defaunation outperformed those achieved by absence of bacterial species. In addition, defaunation was associated with increased propionates at the expense of acetates, while, abscence of bacteria did not alter VFAs molar proportions. Moreover, late stages of incubation were associated with decreased bacterial and protozoal (entodinomorphs and holotrichs) counts, decreased acetic acid and methane production, ‘increased pH value, ammonia nitrogen concentration, propionic .and butyric acids molar proportions. Despite VFAs concentrations and cellulose degradation appeared numerically higher at 24 hours of incubation, the rate of production and degradation in the first 12 hours outperformed late stages of incubation. Thus it was concluded that negative effect of rumen protozoa on fungal cellulolytic activity is greater than that of rumen bacteria