The investigation of fecal bacterial die-off in various agricultural and catchment related matrices remains important because of the growing concern over pathogens in agricultural environments and watercourses. The aim of this research was to investigate the die-off of Escherichia coli within cattle manure (both slurry [liquid mix of excrement and urine produced by housed livestock] and feces), soil and runoff water and to determine if cell numbers would be influenced by the presence of cattle manure within soil and runoff water. E. coli survived better within feces than in slurry; cells within feces declined from 7.5 log10 CFU g-1 to 3.3 log10 CFU g-1 in 76 days. Within slurry, cells fell below levels of detection by day 42. E. coli died off more quickly within manure and slurry than in soil amended with the same fecal material, and declined significantly faster within microcosms when introduced to the soil via sterile water rather than cattle manure. E. coli was found to decline more rapidly within wet (50% moisture w/w), rather than dry (25% moisture w/w), soil. Conversely, in runoff water, die-off of E. coli was increased in the presence of feces. Overall, E. coli survived best in soil incorporated with cattle manure > unincorporated cattle manure > water incorporated with cattle manure. The derived die-off characteristics including half life and decimal reduction times can now provide (i) input for predictive models and (ii) information upon which to consider mitigation strategies associated with both manure and land management.