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In Cambodia, most poultry are raised in backyard flocks with a low level of biosecurity, which increases the risk of spread of infectious diseases. The aim of this study was to evaluate the effectiveness of a practical biosecurity intervention based on affordable basic measures. A cluster randomised trial was conducted in 18 villages in Cambodia from November 2009 to February 2011. Generalised estimating equations were used to test the association between the intervention and mortality rates in flocks of chickens and ducks. Mortality rates in chicken flocks in intervention villages (mean 6.3%, range 3.5–13.8%, per month) were significantly higher than in control villages (mean 4.5%, range 2.0–9.7%, per month; P < 0.01). Mortality rates in duck flocks in intervention villages (mean 4.1%, range 1.9–7.9%, per month) were significantly higher than in control villages (mean 2.8%, range 0.6–8.0%, per month; P < 0.01 …