Over two billion people worldwide lack access to an improved sanitation facility that adequately retains or treats feces. This results in the potential for fecal material containing enteric pathogens to contaminate the environment, including household floors. This study aimed to assess how floor type and sanitation practices impacted the concentration of fecal contamination on household floors. We sampled 189 floor surfaces within 63 households in a peri-urban community in Iquitos, Peru. All samples were analyzed for colony forming units (CFUs) of E. coli, and households were evaluated for their water, sanitation, and hygiene characteristics. Results of multivariate linear regression indicated that households with improved sanitation and cement floors in the kitchen area had reduced fecal contamination to those with unimproved sanitation and dirt floors (Beta: -1.18 log10 E. coli CFU/900 cm2 95% confidence interval [CI]: -1.77, -0.60). Households that did not versus did share their sanitation facility also had less contaminated kitchen floors (Beta: -0.65 log10 E. coli CFU/900 cm2 95% CI: -1.15, -0.16). These findings suggest that the sanitation facilities of a home may impact the microbial load found on floors, contributing to the potential for household floors to serve as an indirect route of fecal pathogen transmission to children.