The present study investigated the diversity and genetic structure of Escherichia coli isolates from 100 septic tanks in the Canberra region, Australia. The physicochemical characteristics of the septic tanks were determined to examine the extent to which environmental factors might influence E. coli prevalence, diversity and population structure. The results of this study indicated that the temperature of the septic tank could explain some of the variation observed in the number of E. coli isolates recovered per septic tank, whereas pH was an important driver of E. coli diversity. Conductivity, pH and household size had a significant impact on E. coli population structure, and household size significantly affected the probability of detecting human-associated E. coli lineages [sequence types (STs) 69, 73, 95 and 131] in septic tanks. Phylogroup A and B1 strains were not randomly distributed among septic tanks, and the strong negative association between them may indicate intraspecific competition. The findings of this study suggest that the combination of environmental factors and intraspecific interactions may influence the distribution and genetic structure of E. coli in the environment.