Red rot of sugarcane incited by Colletotrichum falcatum (Went) is the most destructive disease in Punjab and Sindh. It infects sugarcane and causes huge losses. Twenty six infected sugarcane stem samples were collected from major sugarcane growing districts of Punjab comprising Faisalabad, Jhang, Chiniot and Sargodha for further studies. C. falcatum was isolated from each location and considered as a separate strain. Based on growth on oat meal agar medium, the cultures were assigned to two major groups. Among four strains, strain-285 was found to be more virulent belonged to light type and 394 was least aggressive belonged to dark type respectively in pathogenicity test. Out of twenty, nine varieties/lines exhibited resistant response, three moderately resistant, four moderately susceptible and four showed susceptible response for strain-394 belonged to dark type during first year as compared to second year when nine indicated resistant reaction, two moderately resistant, five moderately susceptible and four exhibited susceptible reaction which presented least disease reaction followed by 234, 1148 and 285 respectively belonged to light type. Maximum growth of strain-285 was observed at temperature 30 °C (8.6 cm), pH 7 (6.8 cm) and oat meal agar medium (8.6 cm) after 10 days followed by 1148, 234 and 394 individually. Among fungicides Mancozeb (95.00 %) at 3 % concentration and Plant extract Azadirachta indica (89.90 %) at S (standard dose) exhibited maximum inhibition of strain-234 on tenth day of incubation as compared to other strains like 1148, 394 and 285 separately. Mancozeb and A. indica expressed significant inhibitory effect against the pathogen and also reduced percentage disease incidence in combined method (Sett treatment + soil treatment) under greenhouse and field conditions respectively. Strain-285 was found to be most aggressive which showed maximum loss in Brix (%), Polarity Reading, Polarity (%), Purity (%), C.C.S % and Sugar Recovery % followed by 1148, 234 and minimum loss expressed in canes inoculated with strain-394 in qualitative parameters of yield of sugarcane varieties/lines and in the estimation of yield losses during 2012-13 as compared to 2011-12 separately.