Present study edge on comparative efficacy and in vitro activity of Bos taurus (cow) urine and dung for controlling red rot disease of sugarcane by determining percentage mycelial growth inhibition (MGI) of Colletotrichum falcatum. The study was carried out on nine isolates of Colletotrichum falcatum obtained from, Department of Biotechnology, Uka Tarsadia University. Results showed the percentage inhibition of mycelial growth suppressed and varied greatly with respect to different isolates and days of incubation and kind of bio matters. In case of cow urine, highest percentage (48.76%) MGI was recorded for CHA8 isolate and minimum (26.08%) MGI was recorded for GAN6 isolate. . Efficacy of cow dung was found to be varied greatly for all C. falcatum isolates. Cow dung showed 47.59% MGI for cfCHA8 while minimum percent (21.14%) MGI was recorded for cfNAV. The percentage mycelial growth inhibition found to be varied with time of incubation. In most cases the maximum inhibition was recorded after five days of incubation. Present research accomplished that both the bio-matters were found effective in controlling the growth of C. falcatum to certain extent. The use of cow urine and dung can be the cost-effective and eco-friendly approach for controlling sugarcane red rot disease.