Different helminths and protozoa are transmitted to humans by oral uptake of environmentally resistant parasite stages after hand-to-mouth contact or by contaminated food and water. The aim of this study was to develop and validate a method for the simultaneous detection of parasite stages from fresh produce (lettuce) by a one-way isolation test kit followed by genetic identification (PCR, sequencing). Three sentinel zoonotic agents (eggs of Toxocara canis, Echinococcus multilocularis and oocysts of Toxoplasma gondii) were used to investigate the practicability and sensitivity of the method. The detection limits (100% positive results) in the recovery experiments were four Toxocara eggs, two E. multilocularis eggs and 18 T. gondii oocysts (in 4/5 replicates). In a field study, helminth DNA was detected in 14 of 157 lettuce samples including Hydatigera taeniaeformis (Syn. Taenia taeniaeformis) (four samples), T. polyacantha (three), T. martis (one), E. multilocularis (two) and Toxocara cati (four). Toxoplasma gondii was detected in six of 100 samples. In vivo testing in mice resulted in metacestode growth in all animals injected with 40–60 E. multilocularis eggs, while infection rates were 20–40% with 2–20 eggs. The developed diagnostic strategy is highly sensitive for the isolation and genetic characterisation of a broad range of parasite stages from lettuce, whereas the sensitivity of the viability tests needs further improvement.