To prevent fresh produce-associated outbreaks, effective hand hygiene is critical, although determining the effectiveness of hand hygiene interventions in the agricultural environment has not been well studied. The primary purpose of this study was to compare the effect of two hand hygiene interventions on the concentrations and presence of soil (A600 nm of hand rinsate) and microbes on the hands of agricultural field workers. We compared hand washing with soap, a two-step alcohol-based hand sanitizer (Two-Step ABHS) intervention, and not conducting hand hygiene (control). Hand rinsates from 159 farm workers, on two produce farms in Nuevo Leon, Mexico during May 2013, were analyzed spectrophotometrically for indicator bacteria (coliforms, Enterococcus, generic Escherichia coli), and for Bacteroidales 16s rDNA. The hand washing with soap group, compared to the control group, had significantly lower amounts of soil on hands (A600 nm 0.005 vs. A600 nm 0.236, p < 0.001), but had no significant differences in concentrations or proportions of indicator bacteria. The Two-Step ABHS group, compared to the control group, had significantly lower amounts of soil (A600 nm 0.099 vs A600 nm 0.236, p < 0.001), significantly lower concentrations of indicator bacteria (geometric mean Log CFU per hand 1.5 vs 3.3 for coliforms, p < 0.001; 3.1 vs 4.1 for Enterococcus, p < 0.05), and a significantly lower proportion of samples positive for coliforms (53% vs 100%, p < 0.001) and the Bacteroidales AllBac marker (69% vs 90%, p < 0.05). The secondary purpose of our study was to evaluate the sustained effect of hand washing and Two-Step ABHS after 30 min of jalapeño harvest. The hand washing group had significantly lower soil compared to the control group, but no group had significantly different amounts of bacteria than the control group. Two-Step ABHS may be useful for hand hygiene in an agricultural field environment, especially when soap and water are not available.