Background: The metropolitan area of Lima, Peru has a third of the nation's population living in slum dwellings that are hypothesized to contribute to inefficient household hygienic practices. The purpose of this study was to quantitatively assess which living conditions have the greatest impact on handwashing practices. Methods: A cross-sectional epidemiological design of participants ≥16 y of age from San Juan de Miraflores, a slum on the outskirts of Lima, Peru, was used. Poisson regression was applied to assess the impact of living conditions on handwashing practices. Results: We could not demonstrate a relationship between living conditions (home structure, overcrowding, water, grey water disposal) and reported handwashing. The reported lack of handwashing is associated with the number of children in the home (those with children <5 y of age were more likely not to report washing their hands) and length of stay in the slum in years. Conclusions: Living conditions play an important role in one's health, therefore improved study designs are needed to determine which strategies are likely to be the most effective in improving outcomes for slum dwellers.