Few studies have detailed the prenatal pesticide exposure levels of women employed in or residing near large-scale agricultural industries. This study reports pesticide metabolite levels during and shortly after pregnancy in a pilot study of workers in Ecuador. Urine samples were collected for 16 rose workers and 10 nonagricultural workers enrolled into the study in early pregnancy. We measured six nonspecific organophosphatedialkylphosphate (DAP) pesticide metabolites, two alkylenebis-dithiocarbamate pesticide metabolites [ethylene thiourea (ETU) and propylene thiourea (PTU)], 3,5,6-trichloro-2-pyridinol (TCPy), malathion dicarboxylic acid, and two pyrethroid metabolites (2,2-dimethylcyclo propanecarboxylic acid and 3-phenooxybenzoic acid). We collected 141 urine samples (mean: 5.4 per woman). We observed high detection frequencies for five DAP metabolites and ETU, PTU, and TCPy. We report elevated levels of ETU in the entire sample (median 4.24 ng/mL, IQR 2.23, 7.18), suggesting other possible non-occupational pathways of exposure. We found no statistical differences in pesticide levels by current employment status, although the highest pesticide levels were among rose workers. We observed within-woman correlation in TCPy and PTU levels, but not in ETU or DAP levels. The present study is the first to characterize prenatal pesticide exposure levels among working women in Ecuador. Limitations include a small sample size and use of a convenience sample. Strengths include a longitudinal design and multiple urine samples per woman. Results provide an initial characterization of prenatal pesticide exposure levels and how these levels vary over pregnancy in a community impacted by agricultural industry and will inform further studies in the region.