Abstract: The Colorado potato beetle, Leptinotarsa decemlineata, is the most widespread and best-known insect pest that causes great economic losses especially on potatoes. As a result of the intensive use of insecticides this species has gradually developed resistance to most pesticides and its regulation is thus currently very difficult. In addition to the use of disputable genetically modified crops as a promising solution to the problem of pest resistance, development of biocontrol methods using natural pathogens might be solution for sustainable potato production. The aim of our study was to assess the efficacy of entomopathogenic fungus Isaria fumosorosea 163-strain CCM 8367 and entomopathogenic nematode Steinernema feltiae strain Ustinov against L. decemlineata under laboratory conditions. Petri dish trials revealed the highest susceptibility in the last-instar larvae followed by pre-pupae and pupae. The median lethal concentration (LC50) of I. fumosorosea was estimated to be 1.03 × 106 blastospores/ml. Simultaneous application of the fungus with the nematodes increased the mortality of L. decemlineata larvae up to 98% and shortened the median lethal time to two days while no obvious changes in development of nematodes in cadavers were found. When, however, nematodes were applied more than 24 hours after fungus treatment, their development was negatively affected and adults were smaller in comparison to control. In soil application experiments, standard soil substrate was inoculated by either I. fumosorosea, S. feltiae or both before last instar larvae, which finished feeding and searched for place to pupate, were placed individually into the pots. The uncorrected mortality of L. decemlineata was 44% and 45% when 1 × 108 blastospores of I. fumosorosea and 1000 IJ of S. feltiae were applied to the pot, respectively. Combined application of the fungus and the nematode at the same doses increased mortality to 84%. We can conclude that both entomopathogens are prospective biocontrol agents against L. decemlineata and that they could be applied together for higher efficacy.