Abstract Background The health and safety of children at schools in Palestine are not paid the attention they require. Our objective was to assess the major risks and patterns associated with hygiene and sanitation at selected schools in Nablus in the West Bank. Methods A two-layer cross-sectional survey was implemented to collect data on hygiene, sanitation, and bacterial contamination from pupils and staff at ten urban schools in Nablus. We took swab samples from 199 children’s and eight food handlers’ dominant hands and from 82 school bathrooms and classrooms. We observed and recorded variables related to school hygiene on a pre-prepared form. Bacterial identification for each sample was done at An-Najah National University. Using univariate and multivariate analysis, risk factors related to hygiene and sanitation were tested for individual and combined association with the presence of bacterial species transmitted primarily through fecal-oral contamination. Approval was obtained from the institutional review board committee at An-Najah University and consent forms were signed by children’s parents and by food handlers. Findings Our findings showed non-statistically significant associations between the presence of bacterial species that indicate a substantial risk of fecal-oral contamination (Listeria, Streptococcus, Microccocus, Bacillus subtilis, and Gram-negative bacilli) and the following factors: school address (univariate analysis, p=0·384), absence of soap in the school, absence of hand washing after use of bathroom, increased number of children per classroom, and decreased numbers of teachers, toilets, and sinks per number of students at school (ordinal logistic regression, p=0·084). Interpretation This pilot study highlights the presence of several risk factors for bacterial contamination, including child behaviour and aspects of the school environment, favouring the possible spread of fecal-oral transmitted diseases. Policy makers’ efforts should be turned towards new policies rendering schools a safer place for Palestinian children. This study has to be validated on a larger scale and in other cities in the occupied Palestinian territories. Funding This research was funded by An-Najah University as a support for research projects of medical students. Contributors RK, GS, and AS conceived the idea for the study. RK wrote the Abstract. DS, HR, and RH collected the samples in Nablus and LK, MA, AA, and FI identified the bacteria. DS, HR, and RH did the analysis under the supervision of RK and GS. MA and LK contributed equally. Declaration of interests We declare no competing interests. Acknowledgments We thank medical laboratory analysis students who assisted in bacterial diagnosis as well to all school directors, teachers, and students in Nablus for making this work possible.