To examine the effects of poor sanitation and hygiene on the prevalence of antimicrobial-resistant bacteria, we surveyed households in two rural and two urban communities in Guatemala (N = 196 randomly selected households). One adult (≥ 18-years old) and, when available, one child (≤ 5 years-old) provided a stool sample. Up to 48 presumptive Escherichia coli isolates were collected from each stool sample (n = 21,256 total) and were subjected to breakpoint assays for ten antibiotics. Mixed-effects logistic models were used to identify potential factors influencing the likelihood of harboring antibiotic-resistant bacteria. For nine out of ten antibiotics, the odds of detecting resistant bacteria decreased by ~ 32% (odds ratios, OR 0.53-0.8, P < 0.001) for every unit of improvement of a hygiene scale. Hygiene differences between households had a greater impact on prevalence compared to antibiotic use differences. The likelihood of detecting resistant isolates was lower for five antibiotics among households that boiled raw milk before consumption (OR 0.31-0.69), and higher for nine antibiotics in urban households (OR > 1.89-9.6). Poor hygiene conditions likely obscure effects of individual antibiotic use, presumably due to enhanced microbial transmission. Consequently, efforts to improve antibiotic stewardship should be coupled with improving hygiene conditions.