Worldwide, various guidelines and directives for human health and the environment aim to reduce anthropogenic mercury (Hg) emission. Forest ecosystems are natural sinks of this element, transferring it from the atmosphere to litter and soil. Millipedes play an important role in litter/soil and may be negatively affected by contamination. As a pioneering contribution to ecotoxicological test guidelines with millipedes in litter, the present study evaluated food preference of millipedes and mercury toxicity, to prevent interference of food types in toxicity responses. Four food preference tests were performed with Trigoniulus corallinus and Rhinocricus padbergi in relation to leaf morphology, leaf fragmentation level and palatability/nutritional quality. Ecotoxicological effects on these animals were evaluated with increasing concentrations of HgCl2 spiked in litter. In both species, Hg concentrations were measured in repugnatorial glands, casts, exoskeleton and gut. In 28day trials, high bioaccumulation factors (BAF) of Hg were found in gut (= 56), exoskeleton (= 49) and casts (= 39) of R. padbergi. BAFs in the gut, exoskeleton and casts of T. corallinus were lower: 52, 45 and 32, respectively. Mortality tests indicated higher sensitivity of T. corallinus to Hg, compared with R. padbergi. Regarding leaf morphology, lanceolate, linear, obovate and ovate leaf shapes and entire margin were preferred by both species. Although higher biomass gain for both species was observed by ingestion of smaller fragmentation stages, T. corallinus preferred more fragmented leaves while R. padbergi consumed more leaves with lower fragmentation level. Both species preferred and grew better in cellulose-rich litter with lower lignin content corresponding to the mixed litter, over single species leaves of Myrceugenia scutellata and Inga edulis. Therefore, future ecological tests with millipedes should consider the use of entire leaves with less irregular shapes, and higher cellulose and lower lignin contents, to provide ideal conditions for millipede growth and activity.