Hepatitis E virus (HEV) can be transmitted from pigs to humans and cause liver inflammation. Pigs are a major reservoir of HEV and most slaughter pigs show evidence of infection by presence of antibodies (ELISA) or viral RNA (PCR). Reducing the number of HEV infected pigs at slaughter would likely reduce human exposure, yet how this can be achieved, is unknown. We aimed to identify and quantify the effect of biosecurity measures to deliver HEV negative batches of pigs to slaughter.A case-control study was performed with Dutch pig farms selected based on results of multiple slaughter batches. Case farms delivered at least one PCR and ELISA negative batch to slaughter (PCR−ELISA−), indicating absence of HEV infection, and control farms had the highest proportion of PCR and/or ELISA positive batches (PCR+ELISA+), indicating high within-farm transmission. Data about biosecurity and housing were collected via a questionnaire and an audit. Variables were selected by regularization (LASSO regression) and ranked, based the frequency of variable selection. The odds ratios (OR) for the relation between case-control status and the highest ranked variables were determined via grouped logistic regression.Thirty-five case farms, with 10 to 60% PCR−ELISA−batches, and 38 control farms with on average 40% PCR+ELISA+batches, were included. Rubber and steel floor material in fattening pens had the highest ranking and increased the odds of a PCR−ELISA−batch by 5.87 (95%CI 3.03–11.6) and 7.13 (95%CI 3.05–16.9) respectively. Cleaning pig driving boards weekly (OR 1.99 (95%CI 1.07–3.80)), and fly control with predatory flies (OR 4.52 (95%CI 1.59–13.5)) were protective, whereas a long fattening period was a risk. This study shows that cleaning and cleanability of floors and fomites and adequate fly control are measures to consider for HEV control in infected farms. Yet, intervention studies are needed to confirm the robustness of these outcomes.