The diamondback moth (Plutella xylostella) is a major pest of broccoli crops in Colombia. To control P. xylostella, we evaluated the interaction of Beauveria bassiana Bb9205 and Metarhizium anisopliae Ma9236 with Heterorhabditis bacteriophora HNI0100 and its bacterial symbiont Photorhabdus luminescens HNI0100. We used antagonism and disk diffusion assays with fungal extracts to test the interaction between symbiotic bacterium and fungi. P. luminescens inhibited the growth of B. bassiana and M. anisopliae up to 40% by the secretion of secondary metabolites, whereas fungal extracts did not inhibit P. luminescens; this explains the in vivo interactions of these biological control agents. To test the interaction between fungi and nematodes, we first inoculated the fungi followed by the nematodes on different days (0, 2, 4, and 6). We identified the type of interaction using the formula by Nishimatsu and Jackson (J Econ Entomol 91:410–418, 1998) and established that on days 0, 2 and 4 there was an antagonistic interaction, while a synergistic interaction occurred on day 6. Therefore, the use of the interaction between H. bacteriophora HNI0100 with M. anisopliae Ma9236 and B. bassiana Bb9205 is an innovative alternative for the control of P. xylostella.